Methods and platforms for drug discovery using induced pluripotent stem cells

ABSTRACT

The present invention involves methods for identifying an agent that corrects a phenotype associated with a health condition or a predisposition for a health condition. The invention also involves methods for identifying a diagnostic cellular phenotype, determining the risk of a health condition in a subject, methods for reducing the risk of drug toxicity in a human subject, and methods for identifying a candidate gene that contributes to a human disease. The invention also discloses human induced pluripotent stem cell lines.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. application Ser. No.12/157,967, filed Jun. 13, 2008, which claims the benefit of U.S.Provisional Application No. 61/040,646, filed Mar. 28, 2008, and whichalso claims the benefit of International Application No.PCT/EP2007/010019, filed Nov. 20, 2007, and which also claims thebenefit of Japanese Application No. JPO-2007-159382, filed Jun. 15,2007; this application also claims the benefit of InternationalApplication No. PCT/IB2008/002540, filed Jun. 13, 2008, InternationalApplication No. PCT/EP2008/005047, filed Jun. 13, 2008, U.S. ProvisionalApplication No. 61/061,592, filed Jun. 13, 2008, and U.S. ProvisionalApplication No. 61/061,594, filed Jun. 13, 2008, all of which are hereinincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The pharmaceutical industry has expended vast technical and financialresources to develop novel therapeutic agents. Yet, the failure rate(more than 90%) for lead compounds remains persistently high. Often,lead drug compounds that meet expectations in preclinical models, suchas inbred animal models, or a small number of cell lines, are toxic orineffective when administered to a human clinical trial patientpopulation. A fundamental deficiency in most current drug developmentefforts is that they do not evaluate candidate drug efficacy andtoxicity in the context of the extreme genetic diversity of the humanpatient population. In other words, in the present drug developmentparadigm, drug efficacy and toxicity are not tested on many, if notmost, of the relevant genotype/phenotype combinations present in thehuman population. Indeed, even after successful trials in a relativelysmall human clinical trial population, unexpected adverse effects can berevealed once these drugs are administered to a broader human patientpopulation.

SUMMARY OF THE INVENTION

The present invention involves methods for identifying an agent thatcorrects a phenotype associated with a health condition or apredisposition for a health condition comprising contacting a firstpopulation of cells from a human induced pluripotent stem cell line, orcells differentiated from the human induced pluripotent stem cell line,with a candidate agent; contacting a second population of cells from ahuman induced pluripotent stem cell line, or cells differentiated fromthe human induced pluripotent stem cell line, with a control agent;wherein the cells in both populations comprise at least one endogenousallele associated with the health condition or predisposition for thehealth condition; assaying the two populations and identifying candidateagents as correcting the phenotype if the first population is closer toa normal phenotype following treatment than the second population. Thecondition may be selected from health conditions such as aneurodegenerative disorder, a neurological disorder, a mood disorder, acardiovascular disease, a metabolic disorder, a respiratory disease, adrug sensitivity condition, an eye disease, an immunological disorder,or a hematological disease. The cells may be differentiated from inducedstem cells to neural stem cells, neurons, cardiomyocytes, hepatic stemcells, or hepatocytes. The phenotype described may be apoptosis,intracellular calcium level, calcium flux, protein kinase activity,enzyme activity, cell morphology, receptor activation, proteintrafficking, intracellular protein aggregation, organellar composition,motility, intercellular communication, protein expression, or geneexpression.

The invention also involves methods for identifying a diagnosticcellular phenotype comprising comparing a set of cells from a subject tocells from a subject free of the health condition wherein both sets ofcells were induced pluripotent stem cells, or were cells differentiatedfrom induced pluripotent stem cells, and wherein the comparison isperformed on a computer. The cells may be differentiated from inducedstem cells to neural stem cells, neurons, cardiomyocytes, hepatic stemcells, or hepatocytes.

The invention also involves methods for determining the risk of a healthcondition in a subject comprising comparing at least one phenotypedetermined in a first set of cells derived from the subject to the atleast one phenotype determined in a second set of cells derived fromsubjects free of the health condition and to the at least one phenotypedetermined in a third set of cells derived from subjects suffering fromthe health condition; and indicating that the subject is at high riskfor the health condition if the at least one phenotype determined in thefirst set of cells is more similar to the at least one phenotypedetermined in the third set of cells than the at least one phenotypedetermined in the second set of cells, wherein the first, second, andthird sets of cells were induced pluripotent stem cells, or were cellsdifferentiated from induced pluripotent stem cells, and wherein thecomparison is performed on a computer.

The invention also involves methods for reducing the risk of drugtoxicity in a human subject, comprising contacting one or more cellsdifferentiated from an induced pluripotent stem cell line generated fromthe subject with a dose of a pharmacological agent, assaying thecontacted one or more differentiated cells for toxicity, and prescribingor administering the pharmacological agent to the subject if, and onlyif, the assay is negative for toxicity in the contacted cells. The cellsdifferentiated from the induced pluripotent stem cell line may behepatocytes, cardiomyocytes, or neurons.

The invention also involves methods for identifying a candidate genethat contributes to a human disease, comprising comparing a global geneexpression profile of cultured human cells of a differentiated cell typefrom a plurality of healthy individuals to a global gene expressionprofile of cultured human cells of the differentiated cell type from aplurality of individuals suffering from the human disease andidentifying one or more genes that have different expression levels ascandidate genes that contribute to the human disease, wherein thecomparison is performed on a computer.

The invention also discloses a human induced pluripotent stem cell linegenerated from a subject diagnosed as suffering from a health condition,or comprising at least one endogenous allele associated with a healthcondition or a predisposition for the health condition. The inventionalso discloses an isolated population of human cells comprising neuralstem cells or neurons from a subject having at least one endogenousallele associated with a neurodegenerative disorder, a neurologicaldisorder, or a mood disorder, or from a subject diagnosed with theneurodegenerative disorder, neurological disorder, or mood disorder. Theinvention also discloses an isolated population of human cellscomprising human cardiac progenitor cells or cardiomyocytes from asubject having at least one endogenous allele associated with acardiovascular disease, or from a subject diagnosed with thecardiovascular disease. The invention also discloses an isolatedpopulation of human cells comprising hepatic stem cells or hepatocytesfrom a subject having at least one endogenous allele associated with adrug sensitivity condition, or from a subject diagnosed with the drugsensitivity condition.

The invention further discloses a panel of genetically diverse humaninduced pluripotent stem cell lines, comprising human inducedpluripotent stem cell lines generated from a plurality of individualseach of which carry at least one polymorphic allele that is unique amongthe plurality of individuals.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic comparison of a traditional drug discovery scheme(left) in which lead compounds are tested against a disease target inheterologous systems (e.g., animal models) prior to testing compoundefficacy and safety in patients versus a new drug discovery paradigm(right) in which lead compounds are first identified based on theirefficacy in correcting a disease-relevant cellular phenotype inpatient-derived, disease-relevant cell types.

FIG. 2 is an overview of an exemplary, non-limiting, scheme for patientiPSC-based disease modeling and drug discovery.

FIG. 3 is an overview of an exemplary, non-limiting, scheme for patientiPSC-based testing of lead drug candidate efficacy and safety in cellsfrom a genetically diverse cohort of patient iPSC lines.

FIG. 4 is an overview of an exemplary, non-limiting, scheme for patientiPSC-based identification of predictive biomarkers for drug efficacy andtoxicity. Such biomarkers are used in, e.g., patient stratification forclinical trials of drug candidates, and also for optimal dosing andsafety of approved therapeutics in specific patients or patientpopulations, which is sometimes referred to as “personalized medicine.”

FIG. 5 (Top Panel) shows photomicrographs of fibroblasts from threeSMN1^(−/−) SMA patients and two SMN1^(−/+) healthy control subjects;(Bottom Panel) shows photomicrographs of iPSC colonies derived from thecorresponding SMA case and control subject fibroblasts illustrated inthe top panel.

FIG. 6 shows photomicrographs of embryoid bodies obtained from the SMAcase and control iPSC lines shown in FIG. 5.

FIG. 7 shows immunofluorescence photomicrographs of staining forectodermal (TuJ1), mesodermal (Desmin), and endodermal (AFP) lineagemarkers in cells differentiated from SM10d iPSCs.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Genetic variations (e.g., polymorphic alleles) within and among humanpatient populations underlie, to a large extent, differences inindividual disposition to diseases, disease manifestation, diseaseseverity, and response to treatment (e.g., to drug treatment). Theprevalent animal and cellular models for human disease and drugdiscovery provide a poor representation of the genotypic/phenotypicspectrum extant in the patient populations to be treated. For example,strains of mice and rats commonly used in drug discovery are highlyinbred, and thus only represent a very narrow range of possiblegenotype/phenotype combinations in mice or rats, let alone humans.Likewise, the relatively small number of human cell lines used for drugscreening may reflect the genotypic/phenotypic scope of the individualsfrom which they were derived, but not that of a genetically diversepopulation. Further, most human cell lines are quite limited in theircapacity to generate or phenocopy specific differentiated cell types(e.g., neurons, cardiomyocytes, and hepatocytes) affected by aparticular health condition. Also, the cell lines are not representativeof cell populations in a subject, since cell lines have been altered toindefinitely replicate. Importantly, in many cases animal models orgenetically modified cell models of disease simply fail to adequatelyrecapitulate the cellular disease phenotypes as they actually occur in ahuman patient's cells. Thus, typical preclinical drug discoverystrategies miss many genotype/phenotypes that are present in the humanpopulation and will have a direct impact on the therapeutic efficacy andtoxicity of a candidate drug compound. A practical consequence of thesefacts is that more often than not lead compounds fail in human clinicaltrials despite successful preclinical testing in animal models andtransformed cell line models, as mentioned above. Ideally, drugscreening and drug target discovery would be performed in biologicalmodels that recapitulate the genetic and phenotypic diversity present ina human patient population and the appropriate disease state at thecellular level, well before the clinical trial stage. These drugdiscovery paradigms are illustrated schematically in FIG. 1. In thetraditional drug discovery model (left), candidate therapeutic agentsare selected for clinical trials in patients based on their action onspecific drug targets and their efficacy/lack of toxicity in animalmodels. In an alternative drug discovery model (right) thedisease-relevant cells derived from patient iPSC lines, as describedherein, are the starting point for identification of lead compoundsbased on their ability to ameliorate a disease-relevant cellularphenotype in patient derived cells.

Accordingly, the present disclosure describes human induced pluripotentstem cell lines from selected individuals (e.g., patients), geneticallydiverse panels of such cell lines, differentiated cells derived fromsuch cell lines, and methods for their use in disease modeling, drugdiscovery, diagnostics, and individualized therapy.

II. Definitions

“Candidate drug compound,” as used herein, refers to any test compoundto be assayed for its ability to affect a functional endpoint. Someexamples of such functional endpoints are ligand binding to a receptor,receptor antagonism, receptor agonism, protein-protein interactions,enzymatic activities, transcriptional responses, etc.

“Correcting” a phenotype, as used herein, refers to altering a phenotypesuch that it more closely approximates a normal phenotype.

“iPSC donor,” as used herein, refers to a subject, e.g. a human patientfrom which one or more induced stem cell lines have been generated.Generally, the genome of an iPSC line corresponds to that of its iPSCdonor.

“Phenomic analysis,” as used herein, refers to the analysis ofphenotypes (e.g., resting calcium level, gene expression profiles,apoptotic index, electrophysiological properties, sensitivity to freeradicals, compound uptake and extrusion, kinase activity, secondmessenger pathway responses) exhibited by a particular type of cell(e.g., cardiomyocytes).

“Phenome,” as used herein refers to the set of phenotypes that issubject and cell-type specific. For example, the phenome of hepatocytesand cardiomyocytes from the same individual will be quite distinct eventhough they share the same genome.

An “endogenous allele,” as used herein, refers to a naturally occurringallele that is native to the genome of a cell, i.e., an allele that isnot introduced by recombinant methodologies.

An “iPSC-derived cell,” as used herein, refers to a cell that isgenerated from an iPSC either by proliferation of the iPSC to generatemore iPSCs, or by differentiation of the iPSC into a different celltype. iPSC-derived cells include cells not differentiated directly froman iPSC, but from an intermediary cell type, e.g., a glial progenitorcell, a neural stem cell, or a cardiac progenitor cell.

A “normal” phenotype, as used herein, refers to a phenotype (e.g.,apoptotic rate, resting calcium level, kinase activity, gene expressionlevel) that falls within a range of phenotypes found in healthyindividuals or that are not associated with (e.g., predictive of) ahealth condition.

III. Induced Stem Cell Lines for Drug Screening and Drug TargetDiscovery

A. Overview

The present disclosure provides human induced pluripotent stem cell(iPSC) lines, panels of stem cell lines, and methods for their use indrug discovery, diagnostic, and therapeutic methods as described indetail below. The induced pluripotent stem cell lines disclosed hereinare characterized by long term self renewal, a normal karyotype, and thedevelopmental potential to differentiate into a wide variety of celltypes (e.g., neurons, cardiomyocytes, and hepatocytes). Inducedpluripotent stem cell lines can be differentiated into cell lineages ofall three germ layers, i.e., ectoderm, mesoderm, and endoderm.

An important nexus exists between a subject (e.g., a patient) and iPSClines generated from that subject. First, all of the genotypes of iPSClines and those of the corresponding subject are identical. Thus,genotype-phenotype correlations, uncovered in one are informative forthe other, and vice versa. Second, differentiated cells (e.g., neurons)derived ex vivo from an iPSC line will exhibit a complete set ofcellular phenotypes (referred to herein as a “phenome”) that are verysimilar, if not identical, to those of differentiated cells in vivo inthe corresponding subject. This point is particularly relevant fordeveloping therapeutics targeted to cells that cannot be routinelyobtained from patients (e.g., neurons, cardiomyocytes, hepatocytes, orpancreatic cells). For example, in the case of a patient suffering froma neurodegenerative disease (e.g., parkinson's disease), dopaminergicneurons, which are typically affected by this condition, can be obtainednon-invasively by differentiating an iPSC line from the subject, and canthen be screened in multiple assays. Thus, iPSC lines provide arenewable source of differentiated cells (e.g., inaccessibledifferentiated cells) in which pathological cellular phenotypes that areassociated with a disease, cell type, and individual may be examined andscreened against test compounds. An exemplary, non-limiting embodimentof this approach to disease modeling and drug discovery is schematicallyillustrated in FIG. 2. iPSC lines and iPSC-derived cells (e.g., motorneurons) are also useful for predicting the efficacy and/or adverse sideeffects of a candidate drug compound in specific individuals or groupsof individuals, as schematically illustrated in FIG. 3. For example,test compounds can be tested for toxicity in hepatocytes differentiatedfrom a genetically diverse panel of induced pluripotent stem cells.Toxicity testing in iPSC-derived hepatocytes can reveal both the overalllikelihood of toxicity of a test compound in a target patientpopulation, and the likelihood of toxicity in specific patients withinthat population.

In effect, iPSC lines and iPSC-derived cells (e.g., pancreatic cells)can serve as “cellular avatars,” that reveal cellular phenotypes thatare disease, cell-type, and subject-specific to the extent thephenotypes are determined or predisposed by the genome. Collectively,panels of patient induced stem cell lines will represent a wide range ofgenotype/phenotype combinations in a patient population. Thus, they areuseful for developing therapeutics that are effective and safe across awide range of the relevant target population, or for determining whichindividuals can be treated effectively and safely with a giventherapeutic agent.

B. Screening and Selection of Subject Samples

Some of the methods described herein utilize induced stem cell lines orpanels of induced stem cell lines derived from subjects that meet one ormore pre-determined criteria. In some cases subjects and cellularsamples from such subjects may be selected for the generation of inducedstem cell lines and panels of induced stem cell lines based on one ormore of such pre-determined criteria. These include, but are not limitedto, the presence or absence of a health condition in a subject (e.g,spinal muscular atrophy, Parkinson's disease, or amyotrophic lateralsclerosis), one or more positive diagnostic criteria for a healthcondition, a family medical history indicating a predisposition orrecurrence of a health condition, the presence or absence of a genotypeassociated with a health condition, or the presence of at least onepolymorphic allele that is not already represented in a panel of inducedstem cell lines.

In some cases, a panel of induced stem cell lines is generatedspecifically from individuals diagnosed with a health condition, andfrom subjects that are free of the health condition. Such healthconditions include, without limitation, neurodegenerative disorders;neurological disorders such as cognitive impairment, and mood disorders;auditory disease such as deafness; osteoporosis; cardiovasculardiseases; diabetes; metabolic disorders; respiratory diseases; drugsensitivity conditions; eye diseases such as macular degeneration;immunological disorders; hematological diseases; kidney diseases;proliferative disorders; genetic disorders, traumatic injury, stroke,organ failure, or loss of limb.

Examples of neurodegenerative disorders include, but are not limited to,Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophiclateral sclerosis, ataxia telangiectasia, Batten disease, bovinespongiform encephalopathy, Canavan disease, Cockayne syndrome,corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,lewy body dementia, Machado-Joseph disease, multiple sclerosis, multiplesystem atrophy, narcolepsy, neuroborreliosis, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, primary lateral sclerosis,prion diseases, Refsum's disease, Sandhoff's disease, Schilder'sdisease, subacute combined degeneration of spinal cord secondary topernicious anaemia, schizophrenia, spinocerebellar ataxia, spinalmuscular atrophy, Steele-Richardson-Olszewski disease, and tabesdorsalis.

Examples of neurological disorders include, stroke, cognitiveimpairment, and mood disorders.

Examples of immunological disorders include but are not limited toacquired immune deficiency, leukemia, lymphoma, hypersensitivities(allergy), autoimmune diseases, and severe combined immune deficiency.

Examples of autoimmune diseases include but are not limited to acutedisseminated encephalomyelitis, addison's disease, ankylosingspondylitis, antiphospholipid antibody syndrome, autoimmune hemolyticanemia, autoimmune hepatitis, bullous pemphigoid, coeliac disease,dermatomyositis, diabetes mellitus type 1, Goodpasture's syndrome,Graves' disease, Guillain-Barré syndrome, Hashimoto's disease,idiopathic thrombocytopenic purpura, lupus erythematosus, multiplesclerosis, myasthenia gravis, pemphigus, pernicious anaemia,polymyositis, primary biliary cirrhosis, rheumatoid arthritis, Sjögren'ssyndrome, temporal arthritis (also known as “giant cell arthritis”),vasculitis, Wegener's granulomatosis.

Examples of cardiovascular diseases include but are not limited toaneurysm, angina, arrhythmia, atherosclerosis, cardiomyopathy,cerebrovascular accident (stroke), cerebrovascular disease, congenitalheart disease, congestive heart failure, myocarditis, valve diseasecoronary, artery disease dilated, cardiomyopathy, diastolic dysfunction,endocarditis, high blood pressure (hypertension), hypertrophiccardiomyopathy, mitral valve prolapse, myocardial infarction (heartattack), and venous thromboembolism.

Examples of metabolic disorders include but are not limited to acidlipase disease, amyloidosis, Barth Syndrome, biotinidase deficiency,carnitine palmitoyl transferase deficiency type II, central pontinemyelinolysis, metabolic diseases of muscle including muscular dystrophy,Farber's Disease, glucose-6-phosphate dehydrogenase deficiency,gangliosidoses, trimethylaminuria, Lesch-Nyhan syndrome, lipid storagediseases, metabolic myopathies, methylmalonic aciduria, mitochondrialmyopathies, mucopolysaccharidoses, mucolipidoses, mucolipidoses,mucopolysaccharidoses, multiple CoA carboxylase deficiency, nonketotichyperglycinemia, Pompe disease, propionic acidemia, type I glycogenstorage disease, urea cycle disorders, hyperoxaluria, and oxalosis.

Examples of proliferative disorders include but are not limited to oneor more of the following: carcinomas, sarcomas, lymphomas, leukemias,germ cell tumors, blastic tumors, prostate cancer, lung cancer,colorectal cancer, bladder cancer, cutaneous melanoma, breast cancer,endometrial cancer, and ovarian cancer.

Further examples of diseases or disorders may be found in U.S.application Ser. No. 12/157,967; filed on Jun. 13, 2008; First InventorKazuhiro Sakurada, 61/061,594; filed on Jun. 13, 2008; First InventorKazuhiro Sakurada, and Ser. No. 12/484,152, filed Jun. 12, 2009; FirstInventor Kazuhiro Sakurada, which are hereby incorporated by reference.It is also anticipated that the methods of the present invention includemarketing and selling products and services for the treatment ofdiseases and disorders including, but not limited to, those mentionedherein.

Such subjects may be identified in, e.g., gene association studies,clinical studies, and hospitals, preferably after a final diagnosis of ahealth condition has been made. Preferably, subjects are identified ingene association studies that include non-affected control individuals.

In other cases, iPSC lines are generated from subjects screened for thepresence or absence of at least one allele associated with a healthcondition or a predisposition for a health condition. Such allelesindicate that an individual, though not exhibiting overt symptoms of ahealth condition, has a high risk of developing the health condition.For example, BRCA1 have been used to indicate a high likelihood ofdeveloping breast cancer. Genotyping of subjects may be performed onsamples from a number of sources, e.g., blood banks, sperm banks,gene-association studies, hospitals, clinical trials, or any othersource as long as a living cellular sample can be obtained from theindividual that is genotyped. While not wishing to be bound by theory,it is believed that one or more that cellular phenotypes fromindividuals carrying alleles associated with health conditions willexhibit abnormalities that can serve as more reliable prognosticindicators of a health condition in combination with a genotype than agenotype alone. Further, identification of specific abnormal cellularphenotypes associated with a health condition may indicate a targetpathway for screening of prophylactic and therapeutic agents for thehealth condition.

There is an ongoing effort to identify associations between polymorphicalleles present in the human population, e.g., single polymorphisms(SNPs) and the occurrence of common health conditions, e.g.,neurodegenerative diseases, psychiatric disorders, metabolic disorders,and cardiovascular diseases. Various types of polymorphic alleles can befound in the human genome as summarized in Table 1.

TABLE 1 Types of Interindividual Variation in the Human Genome GeneticFrequency in change/variation Abbreviation Description human genomeSingle nucleotide SNP Typically two different nucleotides (biallelic12,000,000 polymorphism SNPs) at one defined position, but more rarelyalso triallelic variants occur Deletions/Insertions InDel Deletions (orinsertions, depending on the allele >1,000,000 frequencies) of between 1to 1000 nucleotides. More frequent are deletions of one or threebasepairs Varying number of VNTR Microsatellites also termed shorttandem repeat   >500,000 tandem recaps (STR) polymorphisms are typicallytandem repeats of two, three or four nucleotides, but repeats up to tennucleotides in length may also classified in this group. Minisatellitesare VNTR polymorphisms in which 10-100 nucleotides are repeated invariable numbers. Repeated segments often do not have exactly identicalsequences. VNTRs with larger repeat units (100-1000 bp) are termedsatellites. Copy number CNV Inheritable deletion of multiplication ofDNA >1500 loci variation segments larger than 1 kb. Currently, about1500 covering 12% of CNVs distributed through all chromosomes are thegenome known; estimated to cover 12% of the human genome length.

A number of studies have identified alleles associated with a healthcondition or a predisposition towards a health condition.

Examples of alleles associated with health conditions are known in theart. See, e.g., the databases listed in Table 2.

TABLE 2 List of Publicly Available Databases Containing AllelesAssociated with a Health Condition or Predisposition to a HealthCondition Name of Website Website URL Brief Description Alzgenewww.alzforum.org/res/com/gen/alzgene Collection of published geneticassociation studies performed on Alzheimer Disease phenotypes, fromdatabase searches and journals' contents lists. Case and control datapresented. Cytokine Gene www.nanea.dk/cytokinesnps/ Regularly updateddatabase Polymorphism with Medline-based records in Human from asystematic review of Disease cytokine gene polymorphisms associated withhuman disease. Data extracted from two publications about the study.HuGE Navigator hugenavigator.net HuGE Navigator provides access to acontinuously updated knowledge base in human genome epidemiology,including information on population prevalence of genetic variants,gene-disease associations, gene-gene and gene-environment interactions,and evaluation of genetic tests. GenAtlas www.genatlas.org Regularlyupdated database of genes, phenotypes and references. Among numerousdatabases are brief sections on disorders associated with genes, withlists of citations. May be biased towards statistically significantresults. GeneCanvas genecanvas.idf.inserm.fr Database of cardiovascularcandidate genes and their polymorphisms investigated at INSERM (Paris,France). Data include gene frequencies and linkage disequilibriumstatistics. Genetic geneticassociationdb.nih.gov Database of humangenetic Association association studies of Database complex diseases anddisorders, based on Medline records. Data extracted from publications.Human Obesity obesitygene.pbrc.edu Database of obesity-related Gene Mapgenes, including P values Database for association and references.Biased in favour of statistically significant results. Infeversfmf.igh.cnrs.fr/infevers Database of genetic associations in hereditaryinflammatory disorders, with voluntarily submitted entries. Submissionsare validated by an editorial board member. MedGenemedgene.med.harvard.edu/MEDGENE/ Automated database of gene diseaseassociation studies in Medline. OMIM www.ncbi.nlm.nih.gov/omim/ Databaseof human genes and genetic disorders, containing textual informationwith links to Medline and sequence records in the Entrez system, andlinks to additional related resources at NCBI and elsewhere. PharmGKBwww.pharmgkb.org Database of genomic data and clinical information fromparticipants in pharmacogenetics research studies. Welcomes submissionof primary data. T1DBase t1dbase.org/ Database of type 1 diabetes data,including information from collaborating laboratories. Some indicationgiven of unpublished data.

Some examples of health condition-associated alleles and theircorresponding studies are provided in Table 3.

TABLE 3 Some Examples of Alleles Associated with a Health ConditionPolymorphism(s) Disease identified References Bipolar rs420259 TheWellcome Trust Case Control disorder Consortium (2007), Nature, 447:661-678 Coronary rs1333049 The Wellcome Trust Case Control arteryConsortium (2007), Nature, disease 447: 661-678 Crohn's rs17221417 TheWellcome Trust Case Control disease rs11209026 Consortium (2007),Nature, rs10210302 447: 661-678 rs9858542 rs17234657 rs1000113rs10761659 rs10883365 rs17221417 rs2542151 Hypertension The WellcomeTrust Case Control Consortium (2007), Nature, 447: 661-678 Rheumatoidrs6679677 The Wellcome Trust Case Control arthritis rs6457617 Consortium(2007), Nature, 447: 661-678 Type 1 rs11761231 The Wellcome Trust CaseControl Diabetes rs6679677 Consortium (2007), Nature, rs9272346 447:661-678 rs11171739 rs17696736 rs12708716 Type 2 rs4506565 The WellcomeTrust Case Control Diabetes rs9465871 Consortium (2007), Nature,rs9939609 447: 661-678 Gallstone rs1187534 Bush, et al., (2007), NatGenet, disease (D19H) 39: 995-999 Myocardial rs10757278 Helgadottir, etal., (2007), Science, Infarction 316: 1491-1493 Atrial rs2200733Gudbjartsson, et al., (2007), Nature, fibrillation 448: 353-357 Type 2rs1801282 Warren, et al., (2007) diabetes rs13266634 Pharmacogenomics,7: 180-189 rs1111875 rs7903146 rs5219 rs4402960 rs7754840 rs10811661rs9300039 rs8050136 Type 2 rs13266634 Saxena, et al., (2007), Science,diabetes rs1111875 316: 1331-1336 rs7903146 rs5219 rs1801282 rs10811661rs4402960 rs7754840 Rheumatoid rs3761847 Plenge, et al., (2007), N EnglJ Med, arthritis 357: 1199-209 Exfoliation rs1048661 + Thorleifsson, etal., (2007), Science, Glaucoma rs3825942 317: 1397-1400 Breast rs2981582Easton, et al., (2007), Nature, Cancer rs12443620 447: 1087-1093rs8051542 rs889312 rs3817198 rs2107425 rs13281615 Colorectal rs6983267Tomlinson, et al., (2007), Nat Genet, cancer 39: 984-988

The sequence and other information for any rs-identified SNP can beaccessed on the world wide web through the SNP database of the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/); pulldownmenu=“SNP.”

Subjects may be screened for alleles in genes that affect response to atherapeutic agent or to a class of therapeutic agents. Examples of suchalleles include, but are not limited to, alleles of drug metabolizingenzymes, such as Glucose 6 phosphate dehydrogenase (G6PDH),Butyrlcholine esterase, N-acetyltransferase, Cytochrome P450 isoforms(e.g., 2B6, 2D6, C19, 2C9), Thiopurine S-methyltransferase,Dihydropyrimidine dehydrogenase, and Uridin diphospho-glucuronic acidtransferase type 1A1. Alleles of Cytochrome P450 enzyme isoforms can befound, e.g., in a database provided under the “Home Page of the HumanCytochrome P450 (CYP) Allele Nomenclature Committee,” at“www.cypalleles.ki.se/.” Some alleles occur in genes that affect drugtransport, including, e.g., multiple drug resistance conferringtransporters (MDRs), breast cancer resistance protein (BRCP), multidrugresistance-associated-associated proteins (MRPs), and organicanion-transporting polypeptide (OATP 1B1). Other alleles occur in genesthat encode drug targets, including, but not limited to, Vitamin Kepoxide reductase, Factor V, G-protein coupled receptors (GPCRs). Ofnote, GPCRs are one of the most common drug targets. Examples ofpolymorphic alleles in GPCRs can be found in, e.g., the GPCR NaturalVariants (“NaVa”) Database, which is accessible on the internet at“nava.liacs.nl/” The GPCR NaVa database describes sequence variantswithin the family of human G Protein-Coupled Receptors (GPCRs). Itincludes: rare mutations (frequency<1%); polymorphisms (frequency>1%),including Single Nucleotide Polymorphisms (SNPs); variants withoutestimates of allele frequency.

Polymorphic alleles of interest may be detected and scored in a nucleicacid sample from a subject by any of a number of methods known in theart. For example, detection of multiple alleles may be performed byconducting a nucleic acid array-based assay on a nucleic acid samplefrom a subject, where the nucleic acid array comprises allele-specificprobes (e.g., SNP-specific probes), which, under high stringencyhybridization conditions, selectively hybridize with and discriminatebetween the nucleic acid sequences of two or more polymorphic alleles ofinterest, e.g., alleles of G-protein coupled receptors.

The nucleic acid arrays used to detect polymorphisms may be commerciallyavailable nucleic acid arrays. For example, the Affymetrix® Genome-WideSNP Array 6.0 includes probes for more than 906,000 SNPs and more than946,000 probes for the detection of copy number variation.Alternatively, the nucleic acid arrays may be custom-made to include toa limited subset of alleles of interest. The design of suitable probearrays for analysis of predetermined polymorphisms and interpretation ofthe hybridization patterns is described in detail in WO 95/11995; EP717,113; and WO 97/29212. Such arrays typically contain first and secondgroups of probes which are designed to be complementary to differentallelic forms of the polymorphism. Each group contains a first set ofprobes, which is subdivided into subsets, one subset for eachpolymorphism. Each subset contains probes that span a polymorphism andproximate bases and are complementary to one allelic form of thepolymorphism. Thus, within the first and second probe groups there arecorresponding subsets of probes for each polymorphism. The hybridizationpatterns of these probes to target samples can be analyzed byfootprinting or cluster analysis, as described above. For example, ifthe first and second probes groups contain subsets of probesrespectively complementarity to first and second allelic forms of apolymorphic site spanned by the probes, then on hybridization of thearray to a sample that is homozygous for the first allelic form allprobes in the subset from the first group show specific hybridization,whereas probes in the subset from the second group that span thepolymorphism show only mismatch hybridization. The mismatchhybridization is manifested as a footprint of probe intensities in aplot of normalized probe intensity (i.e., target/reference intensityratio) for the subset of probes in the second group. Conversely, if thetarget sample is homozygous for the second allelic form, a footprint isobserved in the normalized hybridization intensities of probes in thesubset from the first probe group. If the target sample is heterozygousfor both allelic forms then a footprint is seen in normalized probeintensities from subsets in both probe groups although the depression ofintensity ratio within the footprint is less marked than in footprintsobserved with homozygous alleles. Analysis of the hybridization patternof a nucleic acid array to a nucleic acid sample indicates which allelicform is present at some or all of the SNP sequences represented on thearray. Thus, an individual or an iPSC line generated from an individualcan be characterized with a polymorphic profile representing allelicvariants of interest, e.g., alleles associated with a health condition.

In other embodiments, an allele is detected using a primer extensionreaction or amplification reaction. For example, a nucleic acid samplecontaining (or suspected of containing) a target nucleic acid moleculecan be contacted with an oligonucleotide primer that, upon furthercontact with a polymerase, can be extended up to and, if desired, beyondthe position of the SNP. In addition, the nucleic acid sample can becontacted with an amplification primer pair, comprising a first primerand a second primer, which selectively hybridize to complementarystrands of a target nucleic acid molecule and, in the presence ofpolymerase, allow for generation of an amplification product. Forconvenience, the primers of an amplification primer pair are referred toas a “first primer” and a “second primer”; however, reference herein toa “first primer” or a “second primer” is not intended to indicate anyimportance, order of addition, or the like. It will be furtherrecognized that an amplification primer pair requires that the first andsecond primer comprise what are commonly referred to as a forward primerand a reverse primer.

A primer extension or PCR amplification reaction can be designed suchthat the presence of a particular nucleotide at an SNP position can bedetermined by the presence or size of the extension and/or amplificationproduct, in which case the SNP can be determined using a method such asgel electrophoresis, capillary gel electrophoresis, or massspectrometry; or the amplification product can be sequenced to determinethe nucleotide at the SNP position. In addition, the SNP can be detectedindirectly, for example, by further contacting the sample with adetector oligonucleotide, which can selectively hybridize to anucleotide sequence of the first amplification product comprising theSNP position; and detecting selective hybridization of the detectoroligonucleotide, as above.

Various other methods useful for genotyping are known to the art and canbe applied to the present methods. For example, PCR can be performedusing TaqMan® reagents, followed by reading the plates at this endpoint.Molecular beacons, Amplifluor® or TriStar® reagents and methodssimilarly can be used (Stratagene; Intergen). Amplification productsalso can be detected using an ELISA format, for example, using a designin which one primer is biotinylated and the other contains digoxygenin.The amplification products are then bound to a streptavidin plate,washed, reacted with an enzyme-conjugated antibody to digoxygenin, anddeveloped with a chromogenic, fluorogenic, or chemiluminescent substratefor the enzyme. Alternatively, a radioactive method can be used todetect generated amplification products, for example, by including aradiolabeled deoxynucleoside triphosphate into the amplificationreaction, then blotting the amplification products onto DEAE paper fordetection. In addition, if one primer is biotinylated, thenstreptavidin-coated scintillation proximity assay plates can be used tomeasure the PCR products. Additional methods of detection can use achemiluminescent label, for example, a lanthanide chelate such as usedin the DELFIA® assay (Pall Corp.), an electrochemiluminescent label suchas ruthenium tris-bipyridy (ORI-GEN), or a fluorescent label, forexample, using fluorescence correlation spectroscopy.

An assay system that is commercially available and can be used toidentify a nucleotide occurrence of one or more SNPs is the SNP-IT®assay system (Orchid BioSciences, Inc.; Princeton N.J.). In general, theSNP-IT® method is a three step primer extension reaction. In the firststep a target nucleic acid molecule is isolated from a sample byhybridization to a capture primer, which provides a first level ofspecificity. In a second step the capture primer is extended from aterminating nucleotide triphosphate at the target SNP site, whichprovides a second level of specificity. In a third step, the extendednucleotide triphosphate can be detected using a variety of knownformats, including, for example, by direct fluorescence, indirectfluorescence, an indirect colorimetric assay, mass spectrometry, orfluorescence polarization. Reactions conveniently can be processed in384 well format in an automated format using a SNP Stream® instrument(Orchid BioSciences, Inc.).

Various methods for genotyping SNP alleles, selected as describedherein, are readily adaptable to high throughput assays. For example, anamplification reaction such as PCR can be performed using inexpensiverobotic thermocyclers for a specified number of cycles, then theamplification product generated can be determined at the endpoint of thereaction. Furthermore, the methods can be performed in a multiplexformat, for example, using differentially labeled oligonucleotideprobes, or performing oligonucleotide ligation assays that result indifferent sized ligation products, or amplification reactions thatresult in different sized amplification products. In another example,high-throughput mass spectrometry is used to detect SNP alleles in atarget nucleic acid sample. Mass spectrometric methods for SNPgenotyping are described in, e.g., U.S. Pat. Nos. 7,132,519, 6,994,998;and U.S. Patent Application No 20060275789.

Where hybridization-based methods are used, high stringency conditionsare those that result in perfect matches remaining in hybridizationcomplexes, while imperfect matches melt off. Similarly, low stringencyconditions are those that allow the formation of hybridization complexeswith both perfect and imperfect matches. High stringency conditions areknown in the art; see for example Maniatis et al. (1989), MolecularCloning: A Laboratory Manual, 2d Edition; and Short Protocols inMolecular Biology, ed. Ausubel, et al. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993), Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays.” Generally,stringent conditions are selected to be about 5-10 C lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength pH. The Tm is the temperature (under defined ionic strength, pHand nucleic acid concentration) at which 50% of the probes complementaryto the target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 M sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30 C for short probes (e.g.10 to 50 nucleotides) and at least about 60 C for long probes (e.g.greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. In anotherembodiment, less stringent hybridization conditions are used; forexample, moderate or low stringency conditions may be used, as are knownin the art. See, e.g., Maniatis and Ausubel, supra, and Tijssen, supra.

C. Methods for Inducing Pluripotent Stem Cell Lines

iPSC lines may be induced from a wide variety of mammalian cells, e.g.,human somatic cells, such as fibroblasts, bone marrow-derivedmononuclear cells, skeletal muscle cells, adipose cells, peripheralblood mononuclear cells, macrophages, hepatocytes, keratinocytes, oralkeratinocytes, hair follicle dermal cells, gastric epithelial cells,lung epithelial cells, synovial cells, kidney cells, skin epithelialcells or osteoblasts. Methods for inducing multipotent and pluripotentstem cell lines are further disclosed in U.S. application Ser. No.12/157,967; filed Jun. 13, 2008; first inventor Kazuhiro Sakurada,61/061,594; filed on Jun. 13, 2008; First Inventor Kazuhiro Sakurada,and 61/061,565; filed on Jun. 13, 2008; First Inventor KazuhiroSakurada, which are hereby incorporated by reference in their entirety.

The cells to be induced can originate from many different types oftissue, e.g., bone marrow, skin (e.g., dermis, epidermis), muscle,adipose tissue, peripheral blood, foreskin, skeletal muscle, or smoothmuscle. The cells can also be derived from neonatal tissue, including,but not limited to: umbilical cord tissues (e.g., the umbilical cord,cord blood, cord blood vessels), the amnion, the placenta, or othervarious neonatal tissues (e.g., bone marrow fluid, muscle, adiposetissue, peripheral blood, skin, skeletal muscle etc.).

The cells can be derived from neonatal or post-natal tissue collectedfrom a mammal within the period from birth, including cesarean birth, todeath. For example, the tissue may be from a mammal who is >10 minutesold, >1 hour old, >1 day old, >1 month old, >2 months old, >6 monthsold, 1 year old, >2 years old, >5 years old, >10 years old, >15 yearsold, >18 years old, >25 years old, >35 years old, 45 years old, >55years old, >65 years old, <80 years old, <70 years old, <60 years old,<50 years old, <40 years old, <30 years old, <20 years old or <10 yearsold. In some examples, the tissue is from a human age 18, 20, 21, 23,24, 25, 28, 29, 31, 33, 34, 35, 37, 38, 40, 41, 42, 43, 44, 47, 51, 55,61, 63, 65, 70, 77, or 85 years old.

The cells may be from non-embryonic tissue, e.g., at a stage ofdevelopment later than the embryonic stage. In some cases, the cells maybe derived from a fetus. In some cases, the cells are not from a fetus.In some cases, the cells are from an embryo. In some cases, the cellsare not from an embryo.

The cells can be obtained from a single cell or a population of cells.The population may be homogenous or heterogeneous. The cells may be apopulation of cells found in a human cellular sample, e.g., a biopsy orblood sample. In some cases, the cells are a cell line. In some cases,the cells are somatic cells. In some cases, the cells are derived fromcells fused to other cells. In some cases, the cells are not derivedfrom cells fused to other cells. In some cases, the cells are notderived from cells artificially fused to other cells. In some cases, thecells are not: a cell that has been fused with an embryonic stem cell,or a cell that has undergone the procedure known as somatic cell nucleartransfer.

The cellular population may include both differentiated andundifferentiated cells. In some cases, the population primarily containsdifferentiated cells. In other cases, the population primarily containsundifferentiated cells, e.g., undifferentiated stem cells. Theundifferentiated cells within the population may be induced to becomepluripotent or multipotent. In some cases, differentiated cells withinthe cellular population are induced to become pluripotent ormultipotent.

The cellular population may include undifferentiated cells such asmesenchymal stem cells (MSCs), see, e.g., Pittenger et al. (1999),Science 284 (5411): 143-7, multipotent adult progenitor cells (MAPCs),see, e.g., Jahagirdar et al. (2005), Stem Cell Rev. 1(1): 53-9, and/ormarrow-isolated adult multilineage inducible (MIAMI) cells (D'Ippoliotoet al., (2004), J. Cell Sci. 117 (Pt 14): 2971-81. MSCs are multipotentcells that arise from the mesenchyme during development. In some cases,the undifferentiated stem cells (e.g., mesenchymal stem cells, MAPCs andMIAMI cells) are stem cells that have not undergone epigeneticinactivating modification by heterochromatin formation due to DNAmethylation or histone modification of at least four genes, at leastthree genes, at least two genes, at least one gene, or none of thefollowing: Nanog, Oct3/4, Sox2 and Tert. Activation, or expression ofsuch genes, e.g., Tert, Nanog, Oct3/4 or Sox2, may occur when humanpluripotent stem cells are induced from undifferentiated stem cellspresent in a human postnatal tissue.

Methods for obtaining human somatic cells are well established, asdescribed in, e.g., Schantz and Ng (2004), A Manual for Primary HumanCell Culture, World Scientific Publishing Co., Pte, Ltd. In some cases,the methods include obtaining a cellular sample, e.g., by a biopsy,blood draw, or alveolar or other pulmonary lavage. Other suitablemethods for obtaining various types of human somatic cells include, butare not limited to, the following exemplary methods:

Bone Marrow

The donor is given a general anesthetic and placed in a prone position.From the posterior border of the ilium, a collection needle is inserteddirectly into the skin and through the iliac surface to the bone marrow,and liquid from the bone marrow is aspirated into a syringe. Amononuclear cell fraction is then prepared from the aspirate by densitygradient centrifugation. The collected crude mononuclear cell fractionis then cultured prior to use in the methods described herein forinduction pluripotency. For convenience, methods for induction ofpluripotency, as described herein, are collectively referred to as“induction.”

Postnatal Skin

Skin tissue containing the dermis is harvested, for example, from theback of a knee or buttock. The skin tissue is then incubated for 30minutes at 37° C. in 0.6% trypsin/DMEM (Dulbecco's Modified Eagle'sMedium)/F-12 with 1% antibiotics/antimycotics, with the inner side ofthe skin facing downward.

After the skin tissue is turned over to scrub slightly the inner sidewith tweezers, the skin tissue is finely cut into 1 mm2 sections usingscissors, which are then centrifuged at 1200 rpm and room temperaturefor 10 minutes. The supernatant is removed, and to the tissueprecipitate is added 25 ml of 0.1% trypsin/DMEM/F-12/1% antibiotics,antimycotics, and stirred using a stirrer at 37° C. and 200-300 rpm for40 minutes. After confirming that the tissue precipitate is fullydigested, 3 ml fetal bovine serum (FBS) (manufactured by JRH) is added,and filtered sequentially with gauze (Type I manufactured by PIP), a 100μm nylon filter (manufactured by FALCON) and a 40 μm nylon filter(manufactured by FALCON). After centrifuging the resulting filtrate at1200 rpm and room temperature for 10 minutes to remove the supernatant,DMEM/F-12/1% antibiotics, antimycotics is added to wash the precipitate,and then centrifuged at 1200 rpm and room temperature for 10 minutes.The cell fraction thus obtained is then cultured prior to induction.

Postnatal Skeletal Muscle

After the epidermis of a connective tissue containing muscle such as thelateral head of the biceps brachii muscle or the sartorius muscle of theleg is cut and the muscle tissue is excised, it is sutured. The wholemuscle obtained is minced with scissors or a scalpel, and then suspendedin DMEM (high glucose) containing 0.06% collagenase type IA and 10% FBS,and incubated at 37° C. for 2 hours.

By centrifugation, cells are collected from the minced muscle, andsuspended in DMEM (high glucose) containing 10% FBS. After passing thesuspension through a microfilter with a pore size of 40 μm and then amicrofilter with a pore size of 20 μm, the cell fraction obtained may becultured according to the method described in 6. below as crude purifiedcells containing undifferentiated stem cells, and used for the inductionof human pluripotent stem cells of the present invention.

Postnatal Adipose Tissue

Cells derived from adipose tissue for use in the present invention maybe isolated by various methods known to a person skilled in the art. Forexample, such a method is described in U.S. Pat. No. 6,153,432, which isincorporated herein in its entirety. A preferred source of adiposetissue is omental adipose tissue. In humans, adipose cells are typicallyisolated by fat aspiration.

In one method of isolating cells derived from adipose cells, adiposetissue is treated with 0.01% to 0.5%, preferably 0.04% to 0.2%, and mostpreferably about 0.1% collagenase, 0.01% to 0.5%, preferably 0.04%, andmost preferably about 0.2% trypsin and/or 0.5 ng/ml to 10 ng/ml dispase,or an effective amount of hyaluronidase or DNase (DNA digesting enzyme),and about 0.01 to about 2.0 mM, preferably about 0.1 to about 1.0 mM,most preferably 0.53 mM concentration of ethylenediaminetetraacetic acid(EDTA) at 25 to 50° C., preferably 33 to 40° C., and most preferably 37°C. for 10 minutes to 3 hours, preferably 30 minutes to 1 hour, and mostpreferably 45 minutes.

Cells are passed through nylon or a cheese cloth mesh filter of 20microns to 800 microns, more preferably 40 microns to 400 microns, andmost preferably 70 microns. Then the cells in the culture medium aresubjected to differential centrifugation directly or using Ficoll orPercoll or another particle gradient. The cells are centrifuged at 100to 3000×g, more preferably 200 to 1500×g, most preferably 500×g for 1minute to 1 hours, more preferably 2 to 15 minutes and most preferably 5minutes, at 4 to 50° C., preferably 20 to 40° C. and more preferablyabout 25° C.

The adipose tissue-derived cell fraction thus obtained may be culturedaccording to the method described herein as crude purified cellscontaining undifferentiated stem cells, and used for the induction ofhuman pluripotent or multipotent stem cells.

Blood

About 50 ml to about 500 ml vein blood or cord blood is collected, and amononuclear cell fraction is obtained by the Ficoll-Hypaque method, asdescribed in, e.g., Kanof et al. (1993), Current Protocols in Immunology(J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevack, and W.Strober, eds.), ch. 7.1.1.-7.1.5, John Wiley & Sons, New York).

After isolation of the mononuclear cell fraction, approximately 1×10⁷ to1×10⁸ human peripheral blood mononuclear cells are suspended in a RPMI1640 medium containing 10% fetal bovine serum, 100 μg/ml streptomycinand 100 units/ml penicillin, and after washing twice, the cells arerecovered. The recovered cells are resuspended in RPMI 1640 medium andthen plated in a 100 mm plastic petri dish at a density of about 1×10⁷cells/dish, and incubated in a 37° C. incubator at 8% CO₂. After 10 minremaining in suspension are removed and adherent cells are harvested bypipetting. The resulting adherent mononuclear cell fraction is thencultured prior to the induction period as described herein. In somecases, the peripheral blood-derived or cord blood-derived adherent cellfraction thus obtained may be cultured according to the method describedherein as crude purified cells containing undifferentiated stem cells,and used for the induction of human pluripotent stem cells of thepresent invention.

Induction

During the induction process, forced expression of certain polypeptidesis carried out in cultured cells for a period of time, after which theiPSCs are screened for a number of morphological and gene expressionproperties that characterize multipotent and pluripotent stem cells.Induced cells that meet these screening criteria may then be subclonedand expanded. In some cases, the cells to be induced may be cultured fora period of time prior to the induction procedure. Alternatively, thecells to be induced may be used directly in the induction processwithout a prior culture period. In some embodiments, the type of cellculture medium used is the same or very similar before, during, andafter the induction process. In other cases, different cell culturemedia are used at different points. For example, one type of culturemedium may be used directly before the induction process, while a secondtype of media is used during the induction process. At times, a thirdtype of culture medium is used during the induction process.

Cells may be cultured in medium supplemented with a particular serum. Insome embodiments, the serum is fetal bovine serum (FBS). The serum canalso be fetal calf serum (FCS). In some cases, the serum may be Human ABserum. Mixtures of serum may also be used, e.g. mixture of FBS and HumanAB, FBS and FCS, or FCS and Human AB.

Culture of cells may be carried out under a low serum culture conditionsprior to, during, or following induction. A “low serum culturecondition” refers to the use of a cell culture medium containing aconcentration of serum ranging from 0% (v/v) (i.e., serum-free) to about5% (v/v), e.g., 0% to 2%, 0% to 2.5%, 0% to 3%, 0% to 4%, 0% to 5%, 0.1%to 2%, 0.1% to 5%, 0.1%, 0.5%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, or4%. In some embodiments, the serum concentration is from about 0% toabout 2%. In some cases, the serum concentration is about 2%. In somecases, the serum concentration is preferably 2% or less. In otherembodiments, cells are cultured under a “high serum condition,” i.e.,greater than 5% serum to about 20% serum, e.g., 6%, 7%, 8%, 10%, 12%,15%, or 20%. Culturing under high serum conditions may occur prior to,during, and/or after induction.

Some representative media that the cells can be cultured in include:MAPC, FBM, ES, MEF-conditioned ES (MC-ES), and mTeSR™ (available, e.g.,from StemCell Technologies, Vancouver, Canada), See Ludwig et al (2006),Nat Biotechnol, 24(2): 185-187. In other cases, alternative cultureconditions for growth of human ES cells are used, as described in, e.g.,Skottman et al (2006), Reproduction, 132(5):691-698. In someembodiments, the cells are cultured in MAPC, FBM, MC-ES, or mTeSR™ priorto and/or during the introduction of induction factors to the cells; andthe cells are cultured in MC-ES or mTeSR™ medium later in the inductionprocess.

MAPC (2% FBS) Medium may comprise: 60% Dulbecco's Modified Eagle'sMedium-low glucose, 40% MCDB 201, Insulin Transferrin Seleniumsupplement, (0.01 mg/ml insulin; 0.0055 mg/ml transferrin; 0.005 μg/mlsodium selenite), 1× linolenic acid albumin (1 mg/mL albumin; 2 moleslinoneic acid/mole albumin), 1 nM dexamethasone, 2% fetal bovine serum,1 nM dexamethasone, 10-4 M ascorbic acid, and 10 μg/ml gentamycin.

FBM (2% FBS) Medium may comprise: MCDB202 modified medium, 2% fetalbovine serum, 5 μg/ml insulin, 50 mg/ml gentamycin, and 50 ng/mlamphotericin-B.

ES Medium may comprise: 40% Dulbecco's Modified Eagle's Medium (DMEM)40% F12 medium, 2 mM L-glutamine, 1× non-essential amino acids (Sigma,Inc., St. Louis, Mo.), 20% Knockout Serum Replacement™ (Invitrogen,Inc., Carlsbad, Calif.), and 10 μg/ml gentamycin.

MC-ES medium may be prepared as follows. ES medium is conditioned onmitomycin C-treated murine embryonic fibroblasts (MEFs), harvested,filtered through a 0.45-μM filter, and supplemented with about 0.1 mM βmercaptoethanol, about 10 ng/ml bFGF or FGF-2, and, optionally, about 10ng/ml activin A. In some cases, irradiated MEFs are used in place of themitomycin C-treated MEFs.

When either low or high serum conditions are used for culturing thecells, one or more growth factors such as fibroblast growth factor(FGF)-2; basic FGF (bFGF); platelet-derived growth factor (PDGF),epidermal growth factor (EGF); insulin-like growth factor (IGF); orinsulin can be included in the culture medium. Other growth factors thatcan be used to supplement cell culture media include, but are notlimited to one or more: Transforming Growth Factor β-1 (TGF β-1),Activin A, Noggin, Brain-derived Neurotrophic Factor (BDNF), NerveGrowth Factor (NGF), Neurotrophin (NT)-1, NT-2, or NT 3. In some cases,one or more of such factors is used in place of the bFGF or FGF-2 in theMC-ES medium or other cell culture medium.

In some cases, the concentration of growth factors in the culture mediadescribed herein (e.g., MAPC, FBM, MC-ES, mTeSR™) is from about 2 ng/mlto about 20 ng/ml, e.g., about 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6ng/ml, 7 ng/ml, 8 ng/ml, 10 ng/ml, 12 ng/ml, 14 ng/ml, 15 ng/ml, 17ng/ml, or 20 ng/ml. In some embodiments, the concentration of bFGF orFGF2 is from about 2 ng/ml to about 5 ng/ml; from about 5 ng/ml to about8 ng/ml; from about 9 ng/ml to about 11 ng/ml; from about 11 ng/ml toabout 15 ng/ml; or from about 15 ng/ml to about 20 ng/ml.

The growth factors may be used alone or in combination. For example,FGF-2 may be added alone to the medium; in another example, both PDGFand EGF are added to the culture medium.

In some examples, following initiation of the forced expression of genesor polypeptides (e.g., immediately after a retroviral infection period)in cells, the “iPSCs” are maintained in MC-ES medium as describedherein.

In some embodiments, cells are maintained in the presence of a rho, orrho-associated, protein kinase (ROCK) inhibitor to reduce apoptosis. Insome cases, an inhibitor of Rho associated kinase is added to theculture medium. For example, the addition of Y-27632 (Calbiochem; watersoluble) or Fasudil (HA1077: Calbiochem), an inhibitor of Rho associatedkinase (Rho associated coiled coil-containing protein kinase) may beused to culture the human pluripotent stem cells of the presentinvention. In some cases the concentration of Y-27632 or Fasudil, isfrom about 5 μM to about 20 μM, e.g., about 5 μM, 10 μM, 15 μM, or 20μM.

The cells may be cultured for about 1 to about 12 days e.g., 2 days, 3days, 4.5 days, 5 days, 6.5 days, 7 days, 8 days, 9 days, 10 days, orany other number of days from about 1 day to about 12 days prior toundergoing the induction methods described herein.

In some cases, the iPSCs are cultured in complete ES medium in a 37° C.,5% CO₂ incubator, with medium changes about every 1 to 2 days. In someembodiments, induced the iPSCs are cultured and observed for about 14days to about 40 days, e.g., 15, 16, 17, 18, 19, 20, 23, 24, 27, 28, 29,30, 31, 33, 34, 35, 36, 37, 38 days, or any other period from about 14days to about 40 days prior to identifying and selecting clonescomprising “iPSCs” based on morphological characteristics. Morphologicalcharacteristics for identifying iPSC clones include, but are not limitedto, a small cell size with a high nucleus-to-cytoplasm ratio; formationof small monolayer colonies within the space between parental cells(e.g., between fibroblasts).

The cells may be plated at a cell density of about 1×10³ cells/cm² toabout 1×10⁴ cells/cm², e.g., 2×10³ cells/cm², 3.5×10³ cells/cm², 6×10³cells/cm², 7×10³ cells/cm², 9×10³ cells/cm², or any other cell densityfrom about 1×10³ cells/cm² to about 1×10⁴ cells/cm².

The cells can be plated and cultured directly on tissue culture-gradeplastic. Alternatively, cells are plated and cultured on a coatedsubstrate, e.g., a substrate coated with fibronectin, gelatin,Matrigel™, collagen, or laminin. Suitable cell culture vessels include,e.g., 35 mm, 60 mm, 100 mm, and 150 mm cell culture dishes, 6-well cellculture plates, and other size-equivalent cell culture vessels. In somecases, the cells are cultured with feeder cells. For example, the cellsmay be cultured on a layer, or carpet, of MEFs.

Media with low concentrations of serum may be particularly useful toenrich for undifferentiated stem cells. The undifferentiated cellscultured under low serum conditions may or may not share certainproperties with MSCs, MAPCs, and/or MIAMI cells. Differences inphenotype may be due, in part, to culture methods used to obtain MSCs,MAPCs and MIAMI cells. For example, MSCs are often obtained by isolatingthe non-hematopoeitic cells (e.g., interstitial cells) adhering to aplastic culture dish when tissue, e.g., bone marrow, fat, muscle, orskin etc., is cultured in a culture medium containing ahigh-concentration serum (5% or more). However, even under these cultureconditions, a very small number of undifferentiated cells can bemaintained, especially if the cells were passaged under certain cultureconditions (e.g., low passage number or low-density culturing).

In some embodiments, in order to culture and grow human pluripotent stemcells induced from the undifferentiated stem cells of the presentinvention present in a human postnatal tissue, it is preferred that thecells are subcultured every 5 to 7 days in a culture medium containingthe additives described herein on a MEF-covered plastic culture dish ora matrigel-coated plastic culture dish. In some cases, the cells may becultured at a low density, which may be accomplished by splitting thecells from about 1:6 to 1:3 or by plating the cells at 10³ cells/cm² to3×10⁴ cells/cm².

Primary culture ordinarily occurs immediately after the cells areisolated from a donor, e.g., human. The primary cells can be subjectedto a second subculture, a third subculture, a fourth subculture, andgreater than four subcultures. A “second” subculture describes primaryculture cells subcultured once, a “third” subculture describes primarycultures subcultured twice, a “fourth” subculture describes primarycells subcultured three times, etc. The culture techniques describedherein may generally include culturing from the period between theprimary culture and the fourth subculture, but other culture periods mayalso be employed. Preferably, cells are cultured from primary culture tosecond subculture.

Inducing a cell to become pluripotent can be accomplished in numerousways. In some embodiments, the methods for induction of pluripotency inone or more cells include forcing expression of a set of inductionfactors (IFs). In some cases, the set of IFs includes one or more: anOct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide, or a c-Mycpolypeptide. In some cases, the set does not include a c-Mycpolypeptide. For example, the set of IFs can include: an Oct3/4polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide, but not a c-Mycpolypeptide. In some cases, the set of IFs does not include polypeptidesthat might increase the risk of cell transformation.

In some cases, the set may include a c-Myc polypeptide. In certaincases, the c-Myc polypeptide is a constitutively active variant ofc-Myc. In some instances, the set includes a c-Myc polypeptide capableof inducible activity, e.g., a c-Myc-ER polypeptide, see, e.g.,Littlewood, et al. (1995) Nucleic Acid Res. 23(10):1686-90.

In other cases, the set of IFs may include: an Oct3/4 polypeptide, aSox2 polypeptide, and a Klf4 polypeptide, but not a TERT polypeptide, aSV40 Large T antigen polypeptide, HPV16 E6 polypeptide, a HPV16 E7polypeptide, or a Bmi1 polypeptide. In some cases, the set of IFs doesnot include a TERT polypeptide. In some cases, the set of IFs does notinclude a SV40 Large T antigen. In other cases, the set of IFS does notinclude a HPV16 E6 polypeptide or a HPV16 E7 polypeptide.

In some cases, the set of IFs includes three IFs, wherein two of thethree IFs are an Oct3/4 polypeptide and a Sox2 polypeptide. In othercases, the set of IFs includes two IFs, wherein the two polypeptides area c-Myc polypeptide and a Sox2 polypeptide In some cases, the set ofinduction factors is limited to Oct 3/4, Sox2, and Klf4 polypeptides. Inother cases, the set of induction factors may be limited to a set offour IFs: an Oct3/4 polypeptide, a Sox2 polypeptide, a Klf4 polypeptide,and a c-Myc polypeptide.

A set of IFs may include IFs in addition to an Oct 3/4, a Sox2, and aKlf4 polypeptide. Such additional IFs include, but are not limited toNanog, TERT, LIN28, CYP26A1, GDF3, FoxD3, Zfp42, Dnmt3b, Ecat1, and Tcl1polypeptides. In some cases, the set of additional IFs does not includea c Myc polypeptide. In some cases, the set of additional IFs does notinclude polypeptides that might increase the risk of celltransformation.

Forced expression of IFs may be maintained for a period of at leastabout 7 days to at least about 40 days, e.g., 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 25 days, 30 days, 33 days, or 37 days.

The efficiency of inducing pluripotency in cells of a human populationof cells is from at least about 0.001% to at least about 0.01% of thetotal number of cells to be induced, e.g., 0.002%, 0.0034%, 0.004%,0.005%, 0.0065%, 0.007%, 0.008%, or 0.0085%.

D. HDAC Inhibitor

Induction of the cells may be accomplished by combining histonedeacetylase (HDAC) inhibitor treatment with forced expression of sets ofIFs. The cells to be induced may be undifferentiated stem cells presentin a human postnatal tissue. In other cases, the cells to be induced aredifferentiated cells or are a mixture of differentiated orundifferentiated cells.

The HDAC may be combined with the forced expression of a specific set ofIFs, e.g., Oct 3/4, a Sox2, and a Klf4. For example, a human somaticcell is induced to become pluripotent after HDAC inhibitor treatment iscombined with forced expression of Oct3/4, Sox2 and Klf4 or forcedexpression of Oct3/4, Sox2, Klf4, and c-Myc. In some cases, humanpluripotent stem cells can be induced by introducing three genes ofOct3/4, Sox2 and Klf4 or three genes of Oct3/4, Sox2 and Klf4 plus thec-Myc gene or a HDAC inhibitor into undifferentiated stem cells presentin a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4and Sox2 has not undergone epigenetic inactivation. In still othercases, human pluripotent stem cells are induced by introducing threegenes of Oct3/4, Sox2 and Klf4 or three genes of Oct3/4, Sox2 and Klf4plus the c-Myc gene or a histone deacetylase inhibitor intoundifferentiated stem cells after the undifferentiated stem cells wereamplified by a primary culture or a second subculture, or a subculturein a low density and subculturing in a culture medium comprising alow-concentration serum.

Cells may be treated with one or more HDACs for about 2 hours to about 5days, e.g., 3 hours, 6 hours, 12 hours, 14 hours, 18 hours, 1 day, 2days, 3 days, or 4 days. Treatment with HDAC inhibitor may be initiatedprior to beginning forced expression of IFs in the cells. In some cases,HDAC inhibitor treatment begins during or after forced expression of IFsin the cells. In other cases, HDAC inhibitor treatment begins prior toforced expression and is maintained during forced expression.

Suitable concentrations of an HDAC inhibitor range from about 0.001 nMto about 10 mM, depending on the particular HDAC inhibitor to be used,but are selected so as to not significantly decrease cell survival inthe treated cells. The HDAC concentration may range from 0.01 nM, to1000 nM. In some embodiments, the HDAC concentration ranges from about0.01 nM to about 1000 nM, e.g., about 0.05 nM, 0.1 nM, 0.5 nM, 0.75 nM,1.0 nM, 1.5 nM, 10 nM, 20 nM, 40 nM, 50 nM, 100 nM, 200 nM, 300 nM, 500nM, 600 nM, 700 nM, 800 nM, or other concentration from about 0.01 nM toabout 1000 nM. Cells are exposed for 1 to 5 days or 1 to 3 days. Forexample, cells are exposed 1 day, 2 days, 3 days, 4 days or 5 days.

Multiple varieties of HDAC inhibitors can be used for the inductionexperiments. In a preferred embodiment, the HDAC inhibitor MS-275 isused. Examples of suitable HDAC inhibitors include, but are not limitedto, any the following:

A. Trichostatin A and its analogs, for example: trichostatin A (TSA);and trichostatin C (Koghe et al. 1998, Biochem. Pharmacol. 56:1359-1364).

B. Peptides, for example: oxamflatin[(2E)-5-[3-[(phenylsulfonyl)aminophenyl]-pent-2-ene-4-inohydroxamic acid(Kim et al., Oncogene 18: 2461-2470 (1999)); Trapoxin A(cylco-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy-decanoyl)(Kijima et al., J. Biol. Chem. 268: 22429-22435 (1993)); FR901228,depsipeptide (Nakajima et al., Ex. Cell RES. 241: 126-133 (1998));FR225497, cyclic tetrapeptide (H. Mori et al., PCT International PatentPublication WO 00/08048 (Feb. 17, 2000)); apicidin, cyclic tetrapeptide[cyclo-(N—O-metyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al., Proc. Natl. Acad. Sci. U.S.A. 93: 13143-13147(1996); apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, andapicidin IIb (P. Dulski et al., PCT International Patent Publication WO97/11366); HC-toxin, cyclic tetrapeptide (Bosch et al., Plant Cell 7:1941-1950 (1995)); WF27082, cyclic tetrapeptide (PCT InternationalPatent Publication WO 98/48825); and chlamydocin (Bosch et al., supra).

C. Hybrid polar compounds (HPC) based on hydroxamic acid, for example:salicyl hydroxamic acid (SBHA) (Andrews et al., International J.Parasitology 30: 761-8 (2000)); suberoylanilide hydroxamic acid (SAHA)(Richon et al., Proc. Natl. Acad. Sci. U.S.A. 95: 3003-7 (1998));azelaic bishydroxamic acid (ABHA) (Andrews et al., supra);azelaic-1-hydroxamate-9-anilide (AAHA) (Qiu et al., Mol. Biol. Cell 11:2069-83 (2000)); M-carboxy cinnamic acid bishydroxamide (CBHA) (Ricon etal., supra); 6-(3-chlorophenylureido)carpoic hydroxamic acid, 3-Cl-UCHA)(Richon et al., supra); MW2796 (Andrews et al., supra); and MW2996(Andrews et al., supra).

D. Short chain fatty acid (SCFA) compounds, for example: sodium butyrate(Cousens et al., J. Biol. Chem. 254: 1716-23 (1979)); isovalerate(McBain et al., Biochem. Pharm. 53: 1357-68 (1997)); valproic acid;valerate (McBain et al., supra); 4-phenyl butyric acid (4-PBA) (Lea andTulsyan, Anticancer RESearch 15: 879-3 (1995)); phenyl butyric acid (PB)(Wang et al., Cancer RESearch 59: 2766-99 (1999)); propinate (McBain etal., supra); butylamide (Lea and Tulsyan, supra); isobutylamide (Lea andTulsyan, supra); phenyl acetate (Lea and Tulsyan, supra);3-bromopropionate (Lea and Tulsyan, supra); tributyrin (Guan et al.,Cancer RESearch 60: 749-55 (2000)); arginine butyrate; isobutyl amide;and valproate.

E. Benzamide derivatives, for example: MS-275[N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide](Saito et al., Proc. Natl. Acad. Sci. U.S.A. 96: 4592-7 (1999)); and a3′-amino derivative of MS-275 (Saito et al., supra); and CI-994.

A histone deacetylase inhibitor treatment may be carried out, forexample, as follows. The concentration of the HDAC inhibitor may dependon a particular inhibitor, but is preferably 0.001 nM to about 10 mM,and more preferably about 0.01 nM to about 1000 nM. The effective amountor the dosage of a histone deacetylase inhibitor is defined as theamount of the histone deacetylase inhibitor that does not significantlydecrease the survival rate of cells, specifically undifferentiated stemcells. Cells are exposed for 1 to 5 days or 1 to 3 days. The exposureperiod may be less than one day. In a specific embodiment, cells arecultured for about 1 to 5 days, and then exposed to an effective amountof a histone deacetylase inhibitor. However, the histone deacetylaseinhibitor may be added at the start of culturing. Within such a timeframe, a gene-carrying vehicle such as a vector containing a nucleicacid encoding three genes (Oct3/4, Sox2 and Klf4) is introduced intocultured cells by a known method.

E. IF Expression Vectors

Forced expression of the IFs may comprise introducing one or moremammalian expression vectors encoding an Oct 3/4, a Sox2, and a Klf4polypeptide to a population of cells. The IFs may be introduced into thecells as exogenous genes. In some cases, the exogenous genes areintegrated into the genome of a host cell and its progeny. In othercases, the exogenous genes persist in an episomal state in the host celland its progeny. Exogenous genes are genes that are introduced to thecell from an external source. A gene as used herein is a nucleic acidthat includes an open reading frame encoding a polypeptide of interest,e.g., an IF. The gene preferably includes a promoter operably linked toan open reading frame. In some cases, a natural version of the gene mayalready exist in the cell but an additional “exogenous gene” is added tothe cell to induce polypeptide expression.

The one or more mammalian expression vectors may be introduced intogreater than 20% of the total population of cells, e.g., 25%, 30%, 35%,40%, 44%, 50%, 57%, 62%, 70%, 74%, 75%, 80%, 90%, or other percent ofcells greater than 20%. A single mammalian expression vector may containtwo or more of the just-mentioned IFs. In other cases, one or moreexpression vectors encoding an Oct 3/4, Sox2, Klf4, and c Mycpolypeptide are used. In some embodiments, each of the IFs to beexpressed is encoded on a separate mammalian expression vector.

In some cases, the IFs are genetically fused in frame with a transportprotein amino acid sequence, e.g., that of a VP22 polypeptide asdescribed in, e.g., U.S. Pat. Nos. 6,521,455, 6,251,398, and 6,017,735.Such VP22 sequences confer intercellular transport of VP22 fusionpolypeptides from cells that have been transfected with a VP22 fusionpolypeptide expression vector to neighboring cells that have not beentransfected or transduced. See, e.g., Lemken et al (2007), Mol Ther,15(2):310-319. Accordingly, the use of IF-VP22 fusion polypeptides cansignificantly increase the functional efficiency of transfectedmammalian expression vectors in the induction methods described herein.

Examples of suitable mammalian expression vectors include, but are notlimited to: recombinant viruses, nucleic acid vectors, such as plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, humanartificial chromosomes, cDNA, cRNA, and PCR product expressioncassettes. Examples of suitable promoters for driving expression of IFsin include retroviral LTR elements; constitutive promoters such as CMV,HSV1-TK, SV40, EF-1α, β actin; PGK, and inducible promoters, such asthose containing Tet-operator elements. In some cases, one or more ofthe mammalian expression vectors encodes, in addition to an IF, a markergene that facilitates identification or selection of cells that havebeen transfected or infected. Examples of marker genes include, but arenot limited to, fluorescent protein genes, e.g., for EGFP, DS-Red, YFP,and CFP; proteins conferring resistance to a selection agent, e.g., theneoR gene, and the blasticidin resistance gene.

1. Recombinant Viruses

Forced expression of an IF may be accomplished by introducing arecombinant virus carrying DNA or RNA encoding an IF to one or morecells. Additionally, the recombinant virus may carry DNA or RNA encodingmore than 1 IF. This includes multiple copies of a single IF or multipleIFs contained within a single virus. For ease of reference, at times avirus will be referred to herein by the IF it is encoding. For example,a virus encoding an Oct3/4 polypeptide, may be described as an “Oct3/4virus.” In certain cases, a virus may encode more than one copy of an IFor may encode more than one IF, e.g., two IFs, at a time.

Different combinations or sets of recombinant viruses may be introducedto the cells. The set of recombinant viruses may include combinationsincluded in any set of IFs described herein. The set of recombinantviruses may include at least: an Oct3/4 virus, a Sox2 virus, and a Klf4virus. The set of recombinant viruses may be limited to a set of fourrecombinant viruses: an Oct3/4 virus, a Sox2 virus, a Klf4 virus, and ac-Myc virus. In some cases, the set of recombinant viruses is limited toa set of at least: an Oct3/4 virus, a Sox2 virus, a Klf4 virus, and ac-Myc virus. In some cases, the set of recombinant viruses is limited toOct 3/4, Sox2, and Klf4 viruses. The set of recombinant viruses may belimited a set of at least: an Oct3/4 virus, a Sox2 virus, and a Klf4virus. In some cases, the set of recombinant viruses includes threerecombinant viruses, wherein two of the three recombinant viruses are anOct3/4 virus and a Sox2 virus. In still other cases, the set ofrecombinant viruses may be limited to a Sox2 virus and a c-Myc virus.

In some cases, the set of recombinant viruses does not include arecombinant virus that encodes a polypeptide that might increase therisk of cell transformation, e.g., a c-Myc polypeptide. For example, theset of recombinant viruses can include: an Oct3/4 virus, a Sox2 virus,and a Klf4 virus but not a c-Myc virus.

In other cases, the set of recombinant viruses includes a c-Myc virus.The c-Myc polypeptide encoded by the c-Myc virus may be wild-type c-Mycor a constitutively active variant of c-Myc. In some instances, the setincludes a virus encoding c-Myc polypeptide capable of inducibleactivity, e.g., a c-Myc-ER polypeptide, see, e.g., Littlewood, et al.(1995) Nucleic Acid Res. 23(10):1686-90.

The set of recombinant viruses may include: an Oct3/4 virus, a Sox2virus, and a Klf4 virus, but not a TERT virus, a SV40 Large T antigenvirus, HPV16 E6 virus, a HPV16 E7 virus, or a Bmi1 virus. At times, theset of recombinant viruses does not include a TERT virus. In some cases,the set of recombinant viruses does not include a SV40 virus. In othercases, the set of recombinant viruses does not include a HPV16 E6 virusor a HPV16 E7 virus.

A set of recombinant viruses may include viruses in addition to an Oct3/4, a Sox2, and a Klf4 virus. Such additional recombinant virusesinclude, but are not limited to Nanog, TERT, CYP26A1, GDF3, FoxD3,Zfp42, Dnmt3b, Ecat1, and Tcl1 viruses. In some cases, the set ofrecombinant viruses includes any IF variant described herein.

Individual viruses may be added to the cells sequentially in time orsimultaneously. In some cases, at least one virus, e.g., an Oct3/4virus, a Sox2 virus, a Klf4 virus, or a c-Myc virus, is added to thecells at a time different from the time when one or more other virusesare added. In some examples, the Oct3/4 virus, Sox2 virus and KlF4 virusare added to the cells simultaneously, or very close in time, and thec-Myc virus is added at a time different from the time when the otherviruses are added.

At least two recombinant viruses may be added to the cellssimultaneously or very close in time. In some examples, Oct3/4 virus andSox2 virus are added simultaneously, or very close in time, and the Klf4virus or c-Myc virus is added at a different time. In some examples,Oct3/4 virus and Sox2 virus; Oct3/4 virus and Klf4 virus; Oct3/4 virusand c-Myc virus; Sox2 virus and Klf4 virus; Sox2 virus and c-Myc virus;or Klf4 and c-Myc virus are added simultaneously or very close in time.

In some cases, at least three viruses, e.g., an Oct3/4 virus, a Sox2virus, and a Klf4 virus, are added to the cells simultaneously or veryclose in time. In other instances, at least four viruses, e.g., Oct3/4virus, Sox2 virus, Klf4 virus, and c-Myc virus are added to the cellssimultaneously or very close in time.

At times, the efficiency of viral infection can be improved byrepetitive treatment with the same virus. In some cases, one or moreOct3/4 virus, Sox2 virus, Klf4 virus, or c-Myc virus is added to thecells at least two, at least three, or at least four separate times.

Examples of recombinant viruses include, but are not limited, toretroviruses (including lentiviruses); adenoviruses; andadeno-associated viruses. Often, the recombinant retrovirus is murinemoloney leukemia virus (MMLV), but other recombinant retroviruses mayalso be used, e.g., Avian Leukosis Virus, Bovine Leukemia Virus, MurineLeukemia Virus (MLV), Mink-Cell focus-Inducing Virus, Murine SarcomaVirus, Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, MasonPfizer Monkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No.6,333,195.

In other cases, the recombinant retrovirus is a lentivirus (e.g., HumanImmunodeficiency Virus-1 (HIV-1); Simian Immunodeficiency Virus (SIV);or Feline Immunodeficiency Virus (FIV)), See, e.g., Johnston et al(1999), Journal of Virology 73(6)″4991-5000 (FIV); Nègre D et al (2002)Current Topics in Microbiology and Immunology 261:53-74 (SIV); .Naldiniet al (1996) Science. 272:263-267 (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g.,retroviral env) to aid entry into the target cell. Such viralpolypeptides are well-established in the art, see, e.g., U.S. Pat. No.5,449,614. The viral polypeptide may be an amphotropic viralpolypeptide, e.g., amphotropic env, that aids entry into cells derivedfrom multiple species, including cells outside of the original hostspecies. See, e.g., id. The viral polypeptide may be a xenotropic viralpolypeptide that aids entry into cells outside of the original hostspecies. See, e.g., id. In some embodiments, the viral polypeptide is anecotropic viral polypeptide, e.g., ecotropic env, that aids entry intocells of the original host species. See, e.g., id.

Examples of viral polypeptides capable of aiding entry of retrovirusesinto cells include but are not limited to: MMLV amphotropic env, MMLVecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein(VSV-g), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C,FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. Seee.g., Yee et al (1994), Methods Cell Biol. Pt A:99-112 (VSV-G); U.S.Pat. No. 5,449,614. In some cases, the viral polypeptide is geneticallymodified to promote expression or enhanced binding to a receptor.

In general, a recombinant virus is produced by introducing a viral DNAor RNA construct into a producer cell. In some cases, the producer celldoes not express exogenous genes. In other cases, the producer cell is a“packaging cell” comprising one or more exogenous genes, e.g., genesencoding one or more gag, pol, or env polypeptides and/or one or moreretroviral gag, pol, or env polypeptides. The retroviral packaging cellmay comprise a gene encoding a viral polypeptide, e.g., VSV-g that aidsentry into target cells. In some cases, the packaging cell comprisesgenes encoding one or more lentiviral proteins, e.g., gag, pol, env,vpr, vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cellcomprises genes encoding adenovirus proteins such as E1A or E1B or otheradenoviral proteins. For example, proteins supplied by packaging cellsmay be retrovirus-derived proteins such as gag, pol, and env,lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif,tat, rev, and nef; and adenovirus-derived proteins such as E1A and E1B.In many examples, the packaging cells supply proteins derived from avirus that differs from the virus from which the viral vector derives.

Packaging cell lines include but are not limited to anyeasily-transfectable cell line. Packaging cell lines can be based on293T cells, NIH3T3, COS or HeLa cell lines. As packaging cells, anycells may be used that can supply a lacking protein of a recombinantvirus vector plasmid deficient in at least one gene encoding a proteinrequired for virus packaging. Examples of packaging cell lines includebut are not limited to: Platinum-E (Plat-E); Platinum-A (Plat-A); BOSC23 (ATCC CRL 11554); and Bing (ATCC CRL 11270), see, e.g., Morita et al(2000) Gene Therapy 7:1063-1066; Onishi et al (1996) ExperimentalHematology 24:324-329; U.S. Pat. No. 6,995,009. Commercial packaginglines are also useful, e.g., Ampho-Pak 293 cell line, Eco-Pak 2-293 cellline, RetroPack PT67 cell line, and Retro-X Universal Packaging System(all available from Clontech).

The retroviral construct may be derived from a range of retroviruses,e.g., MMLV, HIV-1, SIV, FIV, or other retrovirus described herein. Theretroviral construct may encode all viral polypeptides necessary formore than one cycle of replication of a specific virus. In some cases,the efficiency of viral entry is improved by the addition of otherfactors or other viral polypeptides. In other cases, the viralpolypeptides encoded by the retroviral construct do not support morethan one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In suchcircumstances, the addition of other factors or other viral polypeptidescan help facilitate viral entry. In an exemplary embodiment, therecombinant retrovirus is HIV-1 virus comprising a VSV-g polypeptide butnot comprising a HIV-1 env polypeptide.

The retroviral construct may comprise: a promoter, a multi-cloning site,and/or a resistance gene. Examples of promoters include but are notlimited to CMV, SV40, EF1α, β actin; retroviral LTR promoters, andinducible promoters. The retroviral construct may also comprise apackaging signal (e.g., a packaging signal derived from the MFG vector;a psi packaging signal). Examples of retroviral constructs known in theart include but are not limited to: pMX, pBabeX or derivatives thereof.See e.g., Onishi et al (1996) Experimental Hematology 24:324-329. Insome cases, the retroviral construct is a self-inactivating lentiviralvector (SIN) vector, see, e.g., Miyoshi et al., (1998) J Virol. 72(10):8150-8157. In some cases, the retroviral construct is LL-CG, LS-CG,CL-CG, CS-CG, CLG or MFG. Miyoshi et al., (1998) J Virol. 72(10):8150-8157; Onishi et al (1996) Experimental Hematology 24:324-329;Riviere et al. (1995) PNAS 92: 6733-6737. Virus vector plasmids (orconstructs), include: pMXs, pMXs-IB, pMXs-puro, pMXs-neo (pMXs-IB is avector carrying the blasticidin-resistant gene in stead of thepuromycin-resistant gene of pMXs-puro) [Experimental Hematology, 2003,31 (11): 1007-14], MFG [Proc. Natl. Acad. Sci. U.S.A. 92, 6733-6737(1995)], pBabePuro [Nucleic Acids Research 18, 3587-3596 (1990)], LL-CG,CL-CG, CS-CG, CLG [Journal of Virology 72: 8150-8157 (1998)] and thelike as the retrovirus system, and pAdex1 [Nucleic Acids Res. 23:3816-3821 (1995)] and the like as the adenovirus system. In exemplaryembodiments, the retroviral construct comprises blasticidin (e.g.,pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro); or neomycin (e.g.,pMXs-neo). See, e.g., Morgenstern et al. (1990) Nucleic Acids Research18: 3587-3596.

The retroviral construct may encode one or more IFs. In an exemplaryembodiment, pMX vectors encoding Oct3/4, Sox2, Klf4, or c-Mycpolypeptides, or variants thereof, are generated or obtained. Forexample, Oct3/4 is inserted into pMXs-puro to create pMX-Oct3/4; Sox2 isinserted into pMXs-neo to create pMX-Sox2; Klf4 is inserted into pMXs-IBto create pMX-Klf4; and c-Myc is inserted into pMXs-IB to createpMX-c-Myc.

Methods of producing recombinant viruses from packaging cells and theiruses are well-established, see, e.g., U.S. Pat. Nos. 5,834,256;6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009, incorporatedherein by reference. Many methods begin with the introduction of a viralconstruct into a packaging cell line. The viral construct may beintroduced by any method known in the art, including but not limited to:the calcium phosphate method [Kokai (Japanese Unexamined PatentPublication) No. 2-227075], the lipofection method [Proc. Natl. Acad.Sci. U.S.A. 84: 7413 (1987)], the electroporation method,microinjection, Fugene transfection, and the like, and any methoddescribed herein.

In one example, pMX-Oct3/4, pMX-Sox2, pMX-Klf4 or pMX-c-Myc isintroduced into PlatE cells by Fugene HD (Roche) transfection. The cellculture medium may be replaced with fresh medium comprising FBM (Lonza)supplemented with FGM-2 Single Quots (Lonza). In some embodiments, themedium is replaced from about 12 to about 60 hours following theintroduction of the viral construct, e.g., from about 12 to about 18hours; about 18 to about 24; about 24 to about 30; about 30 to about 36;about 36 to about 42; about 42 to about 48; about 48 to about 54; orabout 54 to about 60 hours following introduction of the viral constructto the producer cells. The medium may be replaced from about 24 to about48 hours after introduction of the viral construct to the producercells. The supernatant can be recovered from about 4 to about 24 hoursfollowing the addition of fresh media, e.g., about 4 hours. In somecases, the supernatant may be recovered about every 4 hours followingthe addition of fresh media. The recovered supernatant may be passedthrough a 0.45 uM filter (Millipore). In some cases, the recoveredsupernatant comprises retrovirus derived from one or more: pMX-Oct3/4,pMX-Sox2, pMX-Klf4 or pMX-c-Myc.

Adenoviral transduction may be used to force expression of the sets ofIFs. Methods for generating adenoviruses and their use are wellestablished as described in, e.g., Straus, The Adenovirus, Plenum Press(NY 1984), 451 496; Rosenfeld, et al, Science, 252:431-434 (1991); U.S.Pat. Nos. 6,203,975, 5,707,618, and 5,637,456. In other cases,adenoviral-associated viral transduction is used to force expression ofthe sets of IFs. Methods for preparing adeno-associated viruses andtheir use are well established as described in, e.g., U.S. Pat. Nos.6,660,514 and 6,146,874.

In an exemplary embodiment, an adenoviral construct is obtained orgenerated, wherein the adenoviral construct, e.g., Adeno-X, comprisesDNA encoding Oct3/4, Sox2, Klf4, or c-Myc. An adenoviral construct maybe introduced by any method known in the art, e.g., Lipofectamine 2000(Invitrogen) or Fugene HD (Roche), into HEK 293 cells. In some cases,the method further comprises (1) collecting the cells when they exhibita cytopathic effect (CPE), such effect occurring from about 10 to about20 days, e.g., about 11, 13, 14, 15, 18, or 20 days after transfection(2) subjecting the cells to from about 2 to about 5 freeze-thaw cycles,e.g., about 3, (3) collecting the resulting virus-containing liquid; (4)purifying the virus using an adenovirus purification kit (Clontech) and(5) storing the virus at −80° C. In some cases, the titer, orplaque-forming unit (PFU), of the adenoviral stocks is determined usingan Adeno-X rapid titer kit (Clontech), as described herein.

The cells may be infected with a recombinant retrovirus that naturallytargets a different cell type or cells originating from a differenthost. To aid infection efficiency, an exogenous receptor may be firstintroduced into the human cells. For example, an exogenous mousereceptor may be added to human cells, e.g., postnatal dermalfibroblasts, in order help entry of murine moloney leukemia virus(MMLV). The exogenous receptor may improve infection efficiency byfacilitating viral entry, especially if the receptor recognizes a viralpolypeptide, e.g., MMLV env, or HIV env. Examples of exogenous receptorsinclude but are not limited to any receptor recognized by a specificretrovirus or lentivirus known in the art. For example, a murinereceptor, mCAT1, GenBank Accession No NM_(—)007513 protein is used inorder to aid MMLV infection of a human target cell. In another example,a CXCR4 or CCR5 receptor is used to aid HIV-1 infection of a targetcell.

The exogenous receptor may be introduced by methods described herein.Methods of introducing the exogenous receptor include but are notlimited to: calcium phosphate transfection, Lipofectamine transfection,Fugene transfection, microinjection, or electroporation. In exemplaryembodiments, a virus, e.g., recombinant adenovirus or retrovirus(including lentivirus), is used to introduce the exogenous receptor tothe target cell. In a further exemplary embodiment, a recombinantadenovirus is used to introduce MCAT1 to human cells and then arecombinant retrovirus, e.g., MMLV, is used to introduce the IF genes,e.g., Oct 3/4, a Sox2, a Klf4, or c-Myc, to the cells.

In some cases, a solution of adenovirus comprising DNA encoding themCAT1 protein, e.g., an adenovirus generated by using a pADEX-mCAT1construct, is generated or obtained. The adenovirus solution cancomprise Hanks' balanced salt solution. In exemplary embodiments,infection of cells is accomplished by: (1) contacting the p-ADEX-mCAT1adenovirus solution with cells, e.g., human, non-embryonic fibroblasts,at a multiplicity of infection (m.o.i.) from about 1:5 to about 1:50,e.g., about 1:5, about 1:7; about 1:10; about 1:15, about 1:20, about1:25; about 1:30, about 1:35; about 1:40; about 1:45, or about 1:50; (2)incubating the cells with the adenovirus solution at room temperaturefrom about 15 minutes to about 2 hours, e.g., about 15 minutes, about 30minutes, about 45 minutes, about 1 hour, about 1.25 hours, about 1.5hours, about 1.75 hours, or about 2 hours; and (3) culturing the somaticcell population in culture medium from about 24 hours to about 60 hours,e.g., about 24 hours, about 30 hours, about 36 hours, about 42 hours,about 48 hours, about 54 hours, or about 60 hours.

The cells can be infected using a wide variety of methods. In somecases, the infection of cells occurs by (1) combining one or more, twoor more, three or more, or all four: pMX-Oct3/4 retrovirus, pMX-Sox2retrovirus, pMX-Klf4, or pMX-c-Myc to obtain a retrovirus solution (2)supplementing the retrovirus solution with from about 2 ug/ml to about15 ug/ml Polybrene, e.g., about 2 ug/ml, about 3 ug/ml, about 5 ug/ml,about 7 ug/ml, about 10 ug/ml, about 12 ug/ml, or about 15 ug/mlPolybrene; (3) contacting the retroviral solution with the somaticcells, at a m.o.i. of from about 1:100 to about 1:500, e.g., about1:100, about 1:150, about 1:200, about 1:250, about 1:300, about 1:350,about 1:400, about 1:450, or about 1:500 m.o.i.; (4) allowing thecontacting of step (3) to continue at 37° C. from about 2 hours to about24 hours, e.g., about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or about 24 hours; (5)soon after the contacting of step (4), changing the medium to MC-ESmedium, as described herein; and (6) changing the MC-ES medium withfresh medium every 1 to 2 days. In some cases, infection of somaticcells occurs by following steps (1) through (6) described herein, withthe added step of pre-incubating the somatic cells for a length of time,e.g., about 48 hours, prior to contacting the cells with the retroviralsolution. Such pre-incubation may be necessary when the somatic cellexpresses an exogenous receptor that was introduced by viraltransduction, transfection, or other method. Thus, in some embodiments,if an adenovirus or lentivirus is used to introduce an exogenousreceptor, e.g., mCAT1, to the somatic cell; such cells may need to becultured for a length of time from at least about 30 hours to at leastabout 60 hours, e.g., about 30, about 35, about 40, about 48, about 52,about 55, or about 60 hours.

The infection of cells may be accomplished by any method known in theart. e.g., Palsson, B., et al. WO95/10619. Apr. 20, 1995; Morling, F. J.et al. (1995). Gene Therapy. 2: 504-508; Gopp et al. (2006) MethodsEnzymol. 420:64-81. For example, the infection may be accomplished byspin-infection or “spinoculation” methods that involve subjecting thecells to centrifugation during the period closely following the additionof virus to the cells. In some cases, virus may be concentrated prior tothe infection, e.g., by ultracentrifugation. In some cases, othertechnologies may be used to aid or improve entry of retroviruses intothe target cell. For example, the retrovirus may be contacted with aliposome or immunoliposome to aid or direct entry into a specific celltype. See, e.g., Tan et al. (2007) Mol Med. 13(3-4): 216-226.

The methods of infecting cells described herein may be used to infectcells expressing an exogenous receptor, e.g., MCAT1 or other exogenousreceptor described herein. Depending on how the exogenous receptor wasintroduced, the preincubation period of the cells prior to infection mayneed to be varied. In some cases, cells that do not express an exogenousreceptor are used. Some recombinant retroviruses, e.g., VSV-Gpseudotyped recombinant retroviruses, may not need the aid of anexogenous receptor in order to efficiently enter cells. In someexamples, VSV-G pseudotyped recombinant retrovirus is introduced tocells following the method described herein, except that the timing ofthe preculturing of the cells may vary.

2. Nucleic Acid Vectors

Nucleic acid vector transfection (e.g., transient transfection) methodsmay be used to introduce IFs into human cells. Methods for preparationof transfection-grade nucleic acid expression vectors are wellestablished. See, e.g., Sambrook and Russell (2001), “Molecular Cloning:A Laboratory Manual,” 3rd ed, (CSHL Press). Examples of high efficiencytransfection efficiency methods include “nucleofection,” as describedin, e.g., Trompeter (2003), J Immunol Methods, 274(1-2):245-256, and ininternational patent application publications WO2002086134, WO200200871,and WO2002086129, transfection with lipid-based transfection reagentssuch as Fugene® 6 and Fugene® HD (Roche), DOTAP, and Lipofectamine™ LTXin combination with the PLUS™ (Invitrogen, Carlsbad, Calif.), Dreamfect™(OZ Biosciences, Marseille, France), GeneJuice™ (Novagen, Madison,Wis.), polyethylenimine (see, e.g., Lungwitz et al (2005), Eur J PharmBiopharm, 60(2):247-266), and GeneJammer™ (Stratagene, La Jolla,Calif.), and nanoparticle transfection reagents as described in, e.g.,U.S. patent application Ser. No. 11/195,066.

3. Protein Transduction

The induction methods may use protein transduction to introduce at leastone of the IFs directly into cells. In some cases, protein transductionmethod includes contacting cells with a composition containing a carrieragent and at least one purified polypeptide comprising the amino acidsequence of one of the above-mentioned IFs. Examples of suitable carrieragents and methods for their use include, but are not limited to,commercially available reagents such as Chariot™ (Active Motif, Inc.,Carlsbad, Calif.) described in U.S. Pat. No. 6,841,535; Bioport® (GeneTherapy Systems, Inc., San Diego, Calif.), GenomeONE (Cosmo Bio Co.,Ltd., Tokyo, Japan), and ProteoJuice™ (Novagen, Madison, Wis.), ornanoparticle protein transduction reagents as described in, e.g., inU.S. patent application Ser. No. 11/138,593.

The protein transduction method may comprise contacting a cells with atleast one purified polypeptide comprising the amino acid sequence of oneof the above-mentioned TAs fused to a protein transduction domain (PTD)sequence (IF-PTD fusion polypeptide). The PTD domain may be fused to theamino terminal of an IF sequence; or, the PTD domain may be fused to thecarboxy terminal of an IF sequence. In some cases, the IF-PTD fusionpolypeptide is added to cells as a denatured polypeptide, which mayfacilitate its transport into cells where it is then renatured.Generation of PTD fusion proteins and methods for their use areestablished in the art as described in, e.g., U.S. Pat. Nos. 5,674,980,5,652,122, and 6,881,825. See also, Becker-Hapak et al (2003), CurrProtocols in Cell Biol, John Wiley & Sons, Inc. Exemplary PTD domainamino acid sequences include, but are not limited to, any of thefollowing:

YGRKKRRQRRR; (SEQ ID NO: 1) RKKRRQRR; (SEQ ID NO: 2) YARAAARQARA; (SEQID NO: 3) THRLPRRRRRR; (SEQ ID NO: 4) and GGRRARRRRRR. (SEQ ID NO: 5)

In some cases, individual purified IF polypeptides are added to cellssequentially at different times. In other embodiments, a set of at leastthree purified IF polypeptides, but not a purified c-Myc polypeptide,e.g., an Oct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptideare added to cells. In some embodiments, a set of four purified IFpolypeptides, e.g., purified Oct3/4, Sox2, Klf4, and c-Myc polypeptidesare added to cells. In some embodiments, the purified IF polypeptidesare added to cells as one composition (i.e., a composition containing amixture of the IF polypeptides). In some embodiments, cells areincubated in the presence of a purified IF polypeptide for about 30minutes to about 24 hours, e.g., 1 hours, 1.5 hours, 2 hours, 2.5 hours,3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12hours, 16 hours, 18 hours, 20 hours, or any other period from about 30minutes to about 24 hours. In some embodiments, protein transduction ofcells is repeated with a frequency of about every day to about every 4days, e.g., every 1.5 days, every 2 days, every 3 days, or any otherfrequency from about every day to about every four days

Forced expression of IFs may also be achieved by using nucleic acid-freeIF-containing protein transducing nanoparticles (PTN). Details ofmethods for generating and using PTNs are found in, e.g., Link et al(2006), Nuc Acids Res, 34(2):e16.

In some cases, the methods described herein utilize protein transductionand expression vector transduction/transfection in any combination toforce expression of a set of IFs as described herein. In someembodiments, retroviral expression vectors are used to force expressionof Oct 3/4, a Sox2, and a Klf4 polypeptides in cells, and purified c-Mycpurified polypeptide is introduced into cells by protein transduction asdescribed herein. HDAC inhibitor treatment can be used in addition tothe purified IF polypeptide. In some cases, a set of at least threepurified IF polypeptides, but not a purified c-Myc polypeptide, e.g., anOct3/4 polypeptide, a Sox2 polypeptide, and a Klf4 polypeptide are addedto cells which are also subjected to HDAC inhibitor treatment.

F. Subcloning Induced Cell Colonies

Cell colonies may be subcloned by any method known in the art. In somecases, the iPSCs are cultured and observed for about 14 days to about 40days, e.g., 15, 16, 17, 18, 19, 20, 23, 24, 27, 28, 29, 30, 31, 33, 34,35, 36, 37, 38 days, or any other period from about 14 days to about 40days prior to identifying and selecting clones comprising “iPSCs” basedon morphological characteristics. Morphological characteristics foridentifying iPSC clones include, but are not limited to, a small cellsize with a high nucleus-to-cytoplasm ratio; formation of smallmonolayer colonies within the space between parental cells (e.g.,between fibroblasts).

After washing cell cultures with a physiological buffer, e.g., Hank'sbalanced salt solution, colonies displaying the morphologicalcharacteristics of interest are surrounded by a cloning ring to thebottom of which silicone grease has been applied. About 100 μl (or 50 μlto 150 μl) of “Detachment Medium For Primate ES Cells” (manufactured byReproCELL, Tokyo Japan) is then added to the cloning ring and incubatedat 37° C. for about 20 minutes to form a cell suspension. The cellsuspension in the ring containing the detached colonies is then added toabout 2 ml of MC ES medium (or other medium described herein), andplated in one well of a MEF-coated 24-well plate or other cell culturevessel of equivalent surface area. After culturing the colony-derivedcells in a 5% CO₂ cell culture incubator at 37° C. for about 14 hours,the medium is replaced. Subsequently, the medium is replaced about everytwo days until about 8 days later when a second subculture is carriedout.

In some embodiments, in the first subculture, the medium is removed, thecells are washed with Hank's balanced salt solution, and DetachmentMedium For Primate ES Cells (ReproCell, Tokyo, Japan) is then added tothe cells and incubated at 37° C. for 10 minutes. After the incubation,MC-ES medium (2 ml) is added to the resulting cell suspension to quenchthe activity of the Detachment Medium. The cell suspension is thentransferred to a centrifuge tube, and centrifuged at 200×g at 4° C. for5 minutes. The supernatant is removed, the cell pellet is resuspended inMC ES medium, and the resuspended cells are plated on four wells of aMEF-coated 24-well plate and cultured for about seven days until asecond subculture is prepared.

In the second subculture, prepared by the method described above, cellsare plated on a 60 mm cell culture dish coated with matrigel at aconcentration of 20 μg/cm². About eight days later (approximately 5weeks after initiating forced expression of IFs), a third subculture isprepared in which cells are plated on two matrigel-coated 60 mm cellculture dishes, one of which can subsequently be used for geneexpression analysis and the other for continued passaging as describedbelow. One of the subcultures is used for gene expression analysis, asdescribed herein, and the other is passaged as needed to maintain a cellline derived from the iPSC clone.

G. Passaging and Maintaining Induced Cells

After subcloning, the iPSCs may be subcultured about every 5 to 7 days.In some cases, the cells are washed with Hank's balanced salt solution,and dispase or Detachment Medium For Primate ES Cells is added, andincubated at 37° C. for 5 to 10 minutes. When approximately more thanhalf of the colonies are detached, MC-ES medium is added to quenchenzymatic activity of the detachment medium, and the resultingcell/colony suspension is transferred to a centrifuge tube. Colonies inthe suspension are allowed to settle on the bottom of the tube, thesupernatant is carefully removed, and MC-ES medium is then added toresuspend the colonies. After examining the size of the colonies, anyextremely large ones are broken up into smaller sizes by slow up anddown pipetting. Appropriately sized colonies are plated on amatrigel-coated plastic culture dish with a base area of about 3 to 6times that before subculture.

Examples of culture media useful for culturing human pluripotent stemcells induced from undifferentiated stem cells present in a humanpostnatal tissue of the present invention include, but are not limitedto, the ES medium, and a culture medium suitable for culturing human EScells such as MEF-conditioned ES medium (MC-ES) or other mediumdescribed herein, e.g., mTeSR™. In some examples, the cells aremaintained in the presence of a ROCK inhibitor, as described herein.

IV. Analysis of Induced Cells

Cell colonies subcultured from those initially identified on the basisof morphological characteristics may be assayed for any of a number ofproperties associated with pluripotent stem cells, including, but notlimited to, expression of alkaline phosphatase activity, expression ofES cell marker genes, expression of protein markers, hypomethylation ofOct3/4 and Nanog promoters relative to a parental cells, long termself-renewal, normal diploid karyotype, and the ability to form ateratoma comprising ectodermal, mesodermal, and endodermal tissues.

A number of assays and reagents for detecting alkaline phosphataseactivity in cells (e.g., in fixed cells or in living cells) are known inthe art. In an exemplary embodiment, colonies to be analyzed are fixedwith a 10% formalin neutral buffer solution at room temperature forabout 5 minutes, e.g., for 2 to 5 minutes, and then washed with PBS. Achromogenic substrate of alkaline phosphatase, 1 step BCIP(5-Bromo-4-Chloro-3′-Indolyphosphate p-Toluidine Salt) and NBT(Nitro-Blue Tetrazolium Chloride) manufactured by Pierce (Rockford,Ill.) is then added and reacted at room temperature for 20 to 30minutes. Cells having alkaline phosphatase activity are stainedblue-violet.

Putative iPS cell colonies tested for alkaline phosphatase activity maybe then assayed for expression of a series of human embryonic stem cellmarker (ESCM) genes including, but not limited to, Nanog, TDGF1, Dnmt3b,Zfp42, FoxD3, GDF3, CYP26A1, TERT, Oct 3/4, Sox2, Sal14, and HPRT. See,e.g., Assou et al (2007), Stem Cells, 25:961-973. Many methods for geneexpression analysis are known in the art. See, e.g., Lorkowski et al(2003), Analysing Gene Expression, A Handbook of Methods: Possibilitiesand Pitfalls, Wiley-VCH. Examples of suitable nucleic acid-based geneexpression assays include, but are not limited to, quantitative RT-PCR(qRT-PCR), microarray hybridization, dot blotting, RNA blotting, RNAseprotection, and SAGE.

In some embodiments, levels of ESCM gene mRNA expression levels inputative iPS cell colonies are determined by qRT-PCR. Putative iPS cellcolonies are harvested, and total RNA is extracted using the “Recoveralltotal nucleic acid isolation kit for formaldehyde- orparaformaldehyde-fixed, paraffin-embedded (FFPE) tissues” (manufacturedby Ambion, Austin, Tex.). In some instances, the colonies used for RNAextraction are fixed colonies, e.g., colonies that have been tested foralkaline phosphatase activity. The colonies can be used directly for RNAextraction, i.e., without prior fixation. In an exemplary embodiment,after synthesizing cDNA from the extracted RNA, the target gene isamplified using the TaqMan® PreAmp mastermix (manufactured by AppliedBiosystems, Foster City, Calif.). Real-time quantitative PCR isperformed using an ABI Prism 7900HT using the following PCR primer sets(from Applied Biosystems) for detecting mRNA of the above-mentioned ESCMgenes: Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_m1, FoxD3,Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT,Hs00162669_m1, GDF3, Hs00220998_m1, CYP26A1, Hs00175627_m1, GAPDH,Hs99999905_m1).

Putative iPS cell colonies may be assayed by an immunocytochemistrymethod for expression of protein markers including, but not limited to,SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, Thy-1, and Nanog. A widerange of immunocytochemistry assays, e.g., fluorescenceimmunocytochemistry assays, are known as described in, e.g., Harlow etal (1988), Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 353-355, and see also, TheHandbook—A Guide to Fluorescent Probes and Labeling Technologies,Molecular Probes, Inc., Eugene, Oreg., (2004).

In an exemplary embodiment, expression of one or more of theabove-mentioned protein markers in putative iPS cell colonies is assayedas follows. Cultured cells are fixed with 10% formaldehyde for 10 minand blocked with 0.1% gelatin/PBS at room temperature for about an hour.The cells are incubated overnight at 4° C. with primary antibodiesagainst SSEA-3 (MC-631; Chemicon), SSEA-4 (MC813-70; Chemicon), TRA-1-60(ab16288; abcam), TRA-1-81 (ab16289; abcam), CD9 (M-L13; R&D systems),CD24 (ALB9; abcam), Thy1 (5E10; BD Bioscience), or Nanog (MAB1997; R&DSystems). For Nanog staining, cells are permeabilized with 0.1% TritonX-100/PBS before blocking. The cell colonies are washed with PBS threetimes, then incubated with AlexaFluor 488-conjugated secondaryantibodies (Molecular Probes) and Hoechst 33258 (Nacalai) at roomtemperature for 1 h. After further washing, fluorescence is detectedwith a fluorescence microscope, e.g., Axiovert 200M microscope (CarlZeiss).

Expression of embryonic stem cell (ESC) marker genes in iPSC coloniesmay be assayed in live cells, which increases the efficiency ofidentifying iPSC colonies following an induction method as describedherein. Examples of ESC marker genes useful for identifying induced stemcell colonies include, e.g., Oct3/4, Nanog, Klf4, Lin28, Sox2, c-Myc, orTERT. In some embodiments, mRNA for one or more of these genes isdetected in live cells. In other embodiments, mRNAs for two or more ofthe ESC marker genes is detected. In one approach, cells are contactedwith one or more molecular beacon probes that hybridize to and signalthe presence of one or more stem cell marker genes. Molecular beacons(MBs) are single-stranded oligonucleotide hybridization probes that forma stem-and-loop structure. The loop contains a probe sequence that iscomplementary to a target sequence, and the stem is formed by theannealing of complementary arm sequences that are located on either sideof the probe sequence. A fluorophore is covalently linked to the end ofone arm and a quencher is covalently linked to the end of the other arm.MBs do not fluoresce when they are free in solution. However, when theyhybridize to a target sequence they undergo a conformational change thatenables them to fluoresce brightly. The probe sequence may range inlength from about 15 to about 30 nucleotides depending on the GC contentof the target probe sequence. Generally, the GC content of the targetprobe sequence should be from about 40 to about 60%. The flanking stemsequences may range from about 5 to about 7 nucleotides with a GCcontent of about 75 to about 100 percent. The design of MBs and theiruse to detect mRNA expression in living cells is known in the art, asdescribed in, e.g., Rhee et al (2008), Nuc Acid Res, 36(5):e30. Usefulalgorithms for determining melting temperatures of an MB duplex and anMB/target duplex are known in the art. See, e.g., the “Mfold” algorithmdescribed in Zucker (2003), Nuc Acids Res 31(13): 3406-3415, which ispublic available on a web server:frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/dna-form1.cgi. Seealso the Hyther Server at: ozone3.chem.wayne.edu/. Typical parametersfor use in these algorithms are 200 nM concentration for beacons andnucleic acid target, a folding temperature of 37° C., and ioniccondition of 10 mM KCl and 5 mM MgCl2. The iPSC colonies to be evaluatedmay be contacted about 14 days to about 50 days after initiatinginduction, e.g., 14 days to 21 days, 14 days to 28 days, 20 days to 45days, 25 days to 40 days, 30 days to 35 days, 30 days to 50 days afterinduction. Preferably, cells are contacted with as low a concentrationof an MB and as short a period as compatible with reliably detecting asignal. In some embodiments, the concentration of an MB of about 0.1 μMto about 5 μM (for each MB), e.g., 0.1 μM to 0.5 μM, 0.2 μM to 1 μM, 0.5μM to 2 μM, or 3 μM to 5 μM. Incubation periods with a MB may range fromabout 5 minutes to about two hours, e.g., 15 minutes to 30 minutes, 20minutes to one hour, 30 minutes to 1.5 hours, 45 minutes to 2 hours, orany other time period form about 5 minutes to two hours. In some cases,MBs are introduced into the cells without the use of a transfectionreagent. In other cases, a transfection reagent optimized foroligonucleotide transfection is utilized, e.g., TransIT® oligotransfection reagent kit or any other transfection reagents known in theart. In other cases, streptolysin-O is used to transiently permealizecells to allow entry of the MBs into the cells. This method is describedin, e.g., Rhee et al supra and Santangelo et al (2004), Nuc Acids Res,32(6): e57.

In some cases, MBs are added to adherent cell cultures and cell coloniesfound to be positive for expression of one or more ESC marker genes arepicked off the substrate as described above. In other cases, MBs areadded to iPSCs in suspension and ESC-positive cells are selected by FACSor any other fluorescence based sorting method. Alternatively, MBs areadded to adherent iPSCs, which are then dispersed prior to FACSselection. Use of FACS for selection of iPSCs is particularly useful forhigh throughput generation of iPSC lines and panels of iPSC lines.

A. Methylation Analysis

In some embodiments, a characteristic of the iPSCs is reducedmethylation of the genomic promoters of Oct3/4 and Nanog relative tothose of their parental cells. Suitable Oct3/4 promoter regions to beanalyzed include, but are not limited to, the Oct3/4 proximal promoterincluding conserved region 1 (CR1) and the Oct3/4 promoter distalenhancer including CR4. Suitable Nanog promoter regions to be analyzedinclude, but are not limited to, the Nanog proximal promoter includingthe Oct3/4 and Sox2 binding sites. See, e.g., Rodda et al (2005), J BiolChem, 280:24731-24737 and Yang et al (2005), J Cell Biochem, 96:821-830.A number of methods for the quantitative analysis of genomic DNA areknown as described in, e.g., Brena et al (2006), J Mol Med,84(5):365-377. In an exemplary embodiment, genomic DNA isolated fromputative iPSCs and cells used for a comparison is isolated and treatedwith bisulfite. Bisulfite-treated genomic DNA is then PCR-amplified withprimers containing a T7 promoter sequence. Afterwards, RNA transcriptsare generated using T7 polymerase and then treated with RNAse A togenerate methylation-specific cleavage products. Methylation ofindividual CpG sites is assessed by MALDI-TOF mass spectrometry of thecleavage products. A detailed description of the method is provided in,e.g., Ehich et al (2005), Proc Natl Acad Sci USA, 102: 15785-15790.

B. Self-Renewal Assay

One of the characteristics of stem cells is their ability to proliferatecontinuously without undergoing senescence. Accordingly, iPSCs areassessed for their ability to be passaged continuously in vitro. In somecases, the iPSCs are assayed for their ability to be passaged for atleast about 30 to at least about 100 times in vitro, e.g., about 33, 35,40, 45, 51, 56, 60, 68, 75, 80, 90, 93, 100, or any other number ofpassages from at least about 30 to at least about 100 passages.

In another evaluation, iPSCs are assayed for their ability toproliferate for a period of about 30 days to about 500 days frominitiation of forced expression of IFs in parental cells, e.g., 40 days,50 days, 60 days, 70 days, 80 days, 100 days, 150 days, 180 days, 200days, 250 days, 300 days, 400 days, 450 days or any other period fromabout 30 days to about 500 days from initiation of forced expression ofIFs in the parental cells. In some embodiments, long-term self-renewalof iPSCs is determined when the cells are passaged in a defined medium(e.g., mTeSR1 medium) and in the absence of feeder cells, e.g., mTeSR1medium as described herein. In other embodiments, cells are passaged inMC-ES medium as described herein.

C. Karyotype Analysis

As another possible analysis, iPSCs are assessed for diploidy and anormal, stable karyotype, e.g., stable after the cells of have beenpassaged for at least one year in vitro. A number of karotype analysismethods are known in the art. In some embodiments, the karyotypeanalysis method is multicolor FISH as described in, e.g., Bayani et al(2004), Curr Protoc Cell Biol, Chapter 22:Unit 22.5. In otherembodiments, the karyotype analysis includes a molecular karyotypeanalysis as described in, e.g., Vermeesch et al (2007), Eur J Hum Genet,15(11):1105-1114. In an exemplary embodiment, iPSCs are pretreated with0.02 μg/ml colecemid for about 2 to about 3 hours, incubated with about0.06 to about 0.075M KCl for about 20 minutes, and then fixed withCarnoy's fixative. Afterwards, for multicolor FISH analysis, cells arehybridized with multicolor FISH probes, e.g., those in the Star*FISH©Human Multicolour FISH (M-FISH) Kit from Cambio, Ltd (Cambridge, UK).

D. Teratoma Analysis

It is generally believed that pluripotent stem cells have the ability toform a teratoma, comprising ectodermal, mesodermal, and endodermaltissues, when injected into an immunocompromised animal. Induced cellsor induced pluripotent stem cells (iPS) or ES cell-like pluripotent stemcells may refer to cells having an in vitro long-term self-renewalability and the pluripotency of differentiating into three germ layers,and said pluripotent stem cells may form a teratoma when transplantedinto a test animal such as mouse.

The iPSCs may be assessed for pluripotency in a teratoma formation assayin an immunocompromised animal model. The immunocompromised animal maybe a rodent that is administered an immunosuppressive agent, e.g.,cyclosporin or FK-506. For example, the immunocompromised animal modelmay be a SCID mouse. About 0.5×10⁶ to about 2.0×10⁶, e.g., 0.6×10⁶,0.8×10⁶, 1.0×10⁶, 1.2×10⁶, 1.5×10⁶, 1.7×10⁶, or other number of iPSCsfrom about 0.5×10⁶ to about 2.0×10⁶ iPSCs/mouse may be injected into themedulla of a testis of a 7- to 8-week-old immunocompromised animal.After about 6 to about 8 weeks, the teratomas are excised afterperfusing the animal with PBS followed by 10% buffered formalin. Theexcised teratomas are then subjected to immunohistological analysis. Onemethod of distinguishing human teratoma tissue from host (e.g., rodent)tissue includes immunostaining for the human-specific nuclear markerHuNu. Immunohistological analysis includes determining the presence ofectodermal (e.g., neuroectodermal), mesodermal, and endodermal tissues.Protein markers for ectodermal tissue include, but are not limited to,nestin, GFAP, and integrin β1. Protein markers for mesodermal tissueinclude, but are not limited to, collagen II, Brachyury, andosteocalcin. Protein markers for endodermal tissue include, but are notlimited to, α-fetoprotein (αFP) and HNF3beta.

E. Global Gene Expression

In some embodiments, global gene expression analysis is performed onputative iPS cell colonies. Such global gene expression analysis mayinclude a comparison of gene expression profiles from a putative iPScell colony with those of one or more cell types, including but notlimited to, (i) parental cells, i.e., one or more cells from which theputative iPS cell colony was induced; (ii) a human ES cell line; or(iii) an established iPS cell line. As known in the art, gene expressiondata for human ES cell lines are available through public sources, e.g.,on the world wide web in the NCBI “Gene Expression Omnibus” database.See, e.g., Barrett et al (2007), Nuc Acids Res, D760-D765. Thus, in someembodiments, comparison of gene expression profiles from a putative iPScolony to those of an ES cell line entails comparison experimentallyobtained data from a putative iPS cell colony with gene expression dataavailable through public databases. Examples of human ES cell lines forwhich gene expression data are publicly available include, but are notlimited to, hE14 (GEO data set accession numbers GSM151739 andGSM151741), Sheff4 (GEO Accession Nos GSM194307, GSM194308, andGSM193409), h_ES 01 (GEO Accession No. GSM194390), h_ES H9 (GEOAccession No. GSM194392), and h_ES BG03 (GEO Accession No. GSM194391).

It is also possible to accomplish global gene expression by analyzingthe total RNA isolated from one or more iPS cell lines by a nucleic acidmicroarray hybridization assay. Examples of suitable microarrayplatforms for global gene expression analysis include, but are notlimited to, the Human Genome U133 plus 2.0 microarray (Affymetrix) andthe Whole Human Genome Oligo Micoarray (Agilent). A number of analyticalmethods for comparison of gene expression profiles are known asdescribed in, e.g., Suarez-Farinas et al (2007), Methods Mol Biol,377:139-152, Hardin et al (2007), BMC Bioinformatics, 8:220, Troyanskayaet al (2002), Bioinformatics, 18(11):1454-1461, and Knudsen (2002), ABiologist's Guide to Analysis of DNA Microarray Data, John Wiley & Sons.In some embodiments, gene expression data from cells produced by themethods described herein are compared to those obtained from other celltypes including, but not limited to, human ES cell lines, parentalcells, and multipotent stem cell lines. Suitable statistical analyticalmetrics and methods include, but are not limited to, the PearsonCorrelation, Euclidean Distance, Hierarchical Clustering (See, e.g.,Eisen et al (1998), Proc Natl Acad Sci USA, 95(25): 14863-14868), andSelf Organizing Maps (See, e.g., Tamayo et al (1999), Proc Natl Acad SciUSA, 96(6):2907-2912.

F. Methods for Differentiating Induced Stem Cell Lines

iPSC lines may be differentiated into cell-types of various lineages.Examples of differentiated cells include any differentiated cells fromectodermal (e.g., neurons and fibroblasts), mesodermal (e.g.,cardiomyocytes), or endodermal (e.g., pancreatic cells) lineages. Thedifferentiated cells may be one or more: pancreatic beta cells, neuralstem cells, neurons (e.g., dopaminergic neurons), oligodendrocytes,oligodendrocyte progenitor cells, hepatocytes, hepatic stem cells,astrocytes, myocytes, hematopoietic cells, or cardiomyocytes.

The differentiated cells derived from the iPSCs may be terminallydifferentiated cells, or they may be capable of giving rise to cells ofa specific lineage. For example, iPSCs can be differentiated into avariety of multipotent cell types, e.g., neural stem cells, cardiac stemcells, or hepatic stem cells. The stem cells may then be furtherdifferentiated into new cell types, e.g., neural stem cells may bedifferentiated into neurons; cardiac stem cells may be differentiatedinto cardiomyocytes; and hepatic stem cells may be differentiated intohepatocytes. Methods for differentiating iPSCs are further disclosed inU.S. application Ser. No. 12/157,967; filed Jun. 13, 2008; firstinventor Kazuhiro Sakurada, 61/061,594; filed on Jun. 13, 2008; FirstInventor Kazuhiro Sakurada, and 61/061,565; filed on Jun. 13, 2008;First Inventor Kazuhiro Sakurada, which are hereby incorporated byreference in their entirety.

There are numerous methods of differentiating the iPSCs into a morespecialized cell type. Methods of differentiating iPSCs may be similarto those used to differentiate other stem cells, particularly ES cells,MSCs, MAPCs, MIAMI, hematopoietic stem cells (HSCs). In some cases, thedifferentiation occurs ex vivo; in some cases the differentiation occursin vivo.

Any known method of generating neural stem cells from ES cells may beused to generate neural stem cells from iPSCs, See, e.g., Reubinoff etal. (2001) Nat Biotechnol. 19(12):1134-40. For example, neural stemcells may be generated by culturing the iPSCs as floating aggregates inthe presence of noggin, or other bone morphogenetic protein antagonist,see e.g., Itsykson et al. (2005) Mol Cell Neurosci. 30(1):24-36. Inanother example, neural stem cells may be generated by culturing theiPSCs in suspension to form aggregates in the presence of growthfactors, e.g., FGF-2, Zhang et al. (2001), Nat. Biotech. (19) 1129-1133.In some cases, the aggregates are cultured in serum-free mediumcontaining FGF-2. In another example, the iPSCs are co-cultured with amouse stromal cell line, e.g., PA6 in the presence of serum-free mediumcomprising FGF-2. In yet another example, the iPSCs are directlytransferred to serum-free medium containing FGF-2 to directly inducedifferentiation.

Neural stems derived from the iPSCs may be differentiated into neurons,oligodendrocytes, or astrocytes. Dopaminergic neurons play a centralrole in Parkinson's Disease and are thus of particular interest. Inorder to promote differentiation into dopaminergic neurons, iPSCs may beco-cultured with a PA6 mouse stromal cell line under serum-freeconditions, see, e.g., Kawasaki et al. (2000) Neuron 28(1):31-40. Othermethods have also been described, see, e.g., Pomp et al. (2005), StemCells 23(7):923-30; U.S. Pat. No. 6,395,546.

Oligodendrocytes may also be generated from the iPSCs. For example,oligodendrocytes may be generated by co-culturing iPSCs or neural stemcells with stromal cells, e.g., Lee et al. (2000) Nature Biotechnol18:675-679. In another example, oligodendrocytes may be generated byculturing the iPSCs or neural stem cells in the presence of a fusionprotein, in which the Interleukin (IL)-6 receptor, or derivative, islinked to the IL-6 cyotkine, or derivative thereof.

Astrocytes may also be produced from the iPSCs. Astrocytes may begenerated by culturing iPSCs or neural stem cells in the presence ofneurogenic medium with bFGF and EGF, see e.g., Brustle et al. (1999)Science 285:754-756.

Induced cells may be differentiated into pancreatic beta cells bymethods known in the art, e.g., Lumelsky et al. (2001) Science292:1389-1394; Assady et al., (2001) Diabetes 50:1691-1697; D'Amour etal (2006) Nat Biotechnol:1392-1401′ D'Amouret al. (2005) Nat Biotechnol23:1534-1541. The method may comprise culturing the iPSCs in serum-freemedium supplemented with Activin A, followed by culturing in thepresence of serum-free medium supplemented with all-trans retinoic acid,followed by culturing in the presence of serum-free medium supplementedwith bFGF and nicotinamide, e.g., Jiang et al. (2007) Cell Res4:333-444. In other examples, the method comprises culturing the iPSCsin the presence of serum-free medium, activin A, and Wnt protein fromabout 0.5 to about 6 days, e.g., about 0.5, 1, 2, 3, 4, 5, 6, days;followed by culturing in the presence of from about 0.1% to about 2%,e.g., 0.2%, FBS and activin A from about 1 to about 4 days, e.g., about1, 2, 3, 4 days; followed by culturing in the presence of 2% FBS,FGF-10, and KAAD-cyclopamine (keto-N-aminoethylaminocaproyl dihydrocinnamoylcyclopamine and retinoic acid from about 1 to about 5 days,e.g., 1, 2, 3, 4, or 5 days; followed by culturing with 1% B27, gammasecretase inhibitor and extendin-4 from about 1 to about 4 days, e.g.,1, 2, 3, or 4 days; and finally culturing in the presence of 1% B27,extendin-4, IGF-1, and HGF for from about 1 to about 4 days, e.g., 1, 2,3, or 4 days.

Hepatic cells or hepatic stem cells may be differentiated from theiPSCs. For example, culturing the iPSCs in the presence of sodiumbutyrate may generate hepatocytes, see e.g., Rambhatla et al. (2003)Cell Transplant 12:1-11. In another example, hepatocytes may be producedby culturing the iPSCs in serum-free medium in the presence of ActivinA, followed by culturing the cells in fibroblast growth factor-4 andbone morphogenetic protein-2, e.g., Cai et al. (2007) Hepatology45(5):1229-39. In an exemplary embodiment, the iPSCs are differentiatedinto hepatic cells or hepatic stem cells by culturing the iPSCs in thepresence of Activin A from about 2 to about 6 days, e.g., about 2, about3, about 4, about 5, or about 6 days, and then culturing the iPSCs inthe presence of hepatocyte growth factor (HGF) for from about 5 days toabout 10 days, e.g., about 5, about 6, about 7, about 8, about 9, orabout 10 days.

The method may also comprise differentiating iPSCs into cardiac musclecells. In an exemplary embodiment, the method comprises culturing theiPSCs in the presence of noggin for from about two to about six days,e.g., about 2, about 3, about 4, about 5, or about 6 days, prior toallowing formation of an embryoid body, and culturing the embryoid bodyfor from about 1 week to about 4 weeks, e.g., about 1, about 2, about 3,or about 4 weeks.

In other examples, cardiomyocytes may be generated by culturing theiPSCs may in the presence of LIF, or by subjecting them to other methodsin the art to generate cardiomyocytes from ES cells, e.g., Bader et al.(2000) Circ Res 86:787-794, Kehat et al. (2001) J Clin Invest108:407-414;; Mummery et al. (2003) Circulation 107:2733-2740.

Examples of methods to generate other cell-types from iPSCs include: (1)culturing iPSCs in the presence of retinoic acid, leukemia inhibitoryfactor (LIF), thyroid hormone (T3), and insulin in order to generateadipoctyes, e.g., Dani et al. (1997) J. Cell Sci 110:1279-1285; (2)culturing iPSCs in the presence of BMP-2 or BMP-4 to generatechondrocytes, e.g., Kramer et al. (2000) Mech Dev 92:193-205; (3)culturing the iPSCs under conditions to generate smooth muscle, e.g.,Yamashita et al. (2000) Nature 408: 92-96; (4) culturing the iPSCs inthe presence of beta-mercaptoethanol to generate keratinocytes, e.g.,Bagutti et al. (1996) Dev Biol 179: 184-196; Green et al. (2003) ProcNatl Acad Sci USA 100: 15625-15630; (5) culturing the iPSCs in thepresence of Interleukin-3 (IL-3) and macrophage colony stimulatingfactor to generate macrophages, e.g., Lieschke and Dunn (1995) Exp Hemat23:328-334; (6) culturing the iPSCs in the presence of IL-3 and stemcell factor to generate mast cells, e.g., Tsai et al. (2000) Proc NatlAcad Sci USA 97:9186-9190; (7) culturing the iPSCs in the presence ofdexamethasone and stromal cell layer, steel factor to generatemelanocytes, e.g., Yamane et al. (1999) Dev Dyn 216:450-458; (8)co-culturing the iPSCs with fetal mouse osteoblasts in the presence ofdexamethasone, retinoic acid, ascorbic acid, beta-glycerophosphate togenerate osteoblasts, e.g., Buttery et al. (2001) Tissue Eng 7:89-99;(9) culturing the iPSCs in the presence of osteogenic factors togenerate osteoblasts, e.g., Sottile et al. (2003) Cloning Stem Cells5:149-155; (10) overexpressing insulin-like growth factor-2 in the iPSCsand culturing the cells in the presence of dimethyl sulfoxide togenerate skeletal muscle cells, e.g., Prelle et al. (2000) BiochemBiophys Res Commun 277:631-638; (11) subjecting the iPSCs to conditionsfor generating white blood cells, e.g., Rathjen et al. (1998) ReprodFertil Dev 10:31-47; or (12) culturing the iPSCs in the presence of BMP4and one or more: SCF, FLT3, IL-3, IL-6, and GCSF to generatehematopoietic progenitor cells, e.g., Chadwick et al. (2003) Blood102:906-915.

In some cases, sub-populations of differentiated cells may be purifiedor isolated. In some cases, one or more monoclonal antibodies specificto the desired cell type are incubated with the cell population andthose bound cells are isolated. In other cases, the desiredsubpopulation of cells expresses a reporter gene that is under thecontrol of a cell type specific promoter.

In a specific embodiment, the hygromycin B phosphotransferase-EGFPfusion protein is expressed in a cell type specific manner. The methodof purifying comprises sorting the cells to select green fluorescentcells and reiterating the sorting as necessary, in order to obtain apopulation of cells enriched for cells expressing the construct (e.g.,hygromycin B phosphotransferase-EGFP) in a cell-type-dependent manner.Selection of desired sub-populations of cells may also be accomplishedby negative selection of proliferating cells with the herpes simplexvirus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system or bypositive selection of cells expressing a bicistronic reporter, e.g.,Anderson et al. (2007) Mol Ther. (11):2027-2036.

G. Panels of Induced Stem Cell Lines

In some cases, the methods described herein utilize a panel of iPSClines or a panel of cells differentiated from iPSC lines. A panel ofiPSC lines comprises multiple iPSC lines, e.g., iPSC lines, that meetcertain selection criteria. Also provided herein are panels of cellsdifferentiated from iPSC lines as described herein. Such panels ofdifferentiated cells include, but are not limited to, panels of neuralstem cells, neurons, retinal cells, glial progenitor cells, glial cells,cardiac progenitor cells, cardiomyocytes, pancreatic progenitor cells,pancreatic beta cells, hepatic stem cells, hepatocytes or lungprogenitor cells. In some cases, the selection criteria for inclusion ofan iPSC line in a panel of iPSC lines are determined prior to generatingthe iPSC lines that will constitute the panel. In other cases, theselection criteria are applied to iPSC lines generated before hand,e.g., a bank of iPSC lines. Selection criteria include, but are notlimited to, the presence or absence of a particular health condition inan iPSC donor, a positive drug response in an iPSC donor, negative,positive, or adverse drug responses in an iPSC donor, the presence orabsence of a particular phenotype in an iPSC line or in cellsdifferentiated from the iPSC line, and the presence or absence of one ormore polymorphic alleles in the cell lines or their correspondingdonors.

In some embodiments, where selection criteria include the presence orabsence of one or more polymorphic alleles, the panel includesgenetically diverse human iPSC lines in which each iPSC line carries atleast one polymorphic allele that is unique among the iPSCs to beincluded in the panel, e.g., 5 to 10, 20 to 50, 50 to 200, 200 to 500,500 to 1000, 1000 to 5000, 5000 to 20000, or 20000 to 50000 polymorphicalleles that are unique within the panel of iPSC lines. Such polymorphicalleles may include, e.g., a SNP allele, a promoter allele, or aprotein-encoding allele. Polymorphic alles can be screened and scoredfor by genotyping using any of a number of known genotyping assays. Insome cases, the genotyping assay is a multiplexed genotyping assay,e.g., a nucleic acid microarray assay platform such as a “SNP chip.” Insome cases, the one or more polymorphic alleles are pre-selected. Insome embodiments, the one or more preselected alleles are polymorphicalleles associated with a health condition or a predisposition to ahealth condition. Examples of polymorphic alleles associated with ahealth condition or a predisposition to a health condition, include, butare not limited to, polymorphic alleles associated with aneurodegenerative disorder, a neurological disorder, an eye disease, amood disorder, a respiratory disease, a cardiovascular disease, animmunological disorder, a hematological disease, a metabolic disorder,or a drug sensitivity condition. Some examples of polymorphic allelesassociated with a health condition are provided in Table 3 above.Polymorphic alleles may include polymorphic alleles in an encodedprotein or a regulatory sequence affecting the expression of the encodedprotein. In some cases, the encoded protein is a drug target. Examplesof drug target proteins include, but are not limited to, GPCRs, ionchannels, kinases, enzymes, and transcription factors.

In other embodiments, the one or more polymorphic alleles arepre-selected based on the presence of a high degree of surroundinglinkage disequilibrium in the genome, which has been proposed as asignature of genomic loci that are likely to impact many common healthconditions. Methods for identifying SNPs having a high surroundinglinkage disequilibrium and genes near such SNPs are described in, e.g.,Wang et al (2006), Proc Natl Acad Sci USA, 103(1):135-140.

In some cases, a panel of iPSC lines includes iPSC lines generated fromsubjects that are diagnosed as suffering from one or more healthconditions. The one or more health conditions may be one or more healthconditions that are common to all of the iPSC donors, or they may behealth conditions that are different between the iPSC donors.

In certain cases, a panel of iPSC lines includes iPSC lines generatedfrom subjects that are both diagnosed as suffering from a healthcondition and carry a polymorphic allele associated with a healthcondition, e.g., a polymorphic allele associated with the diagnosedhealth condition.

A panel of iPSC lines may include iPSC lines from at least about 10individuals to at least about 50,000 individuals, e.g., 10 to 50, 20 to100, 50 to 250, 100 to 1000, 250, to 2000, 500 to 5000, 1000 to 10,000,2500 to 20,000, 10,000, to 30,000, 20,000 to 40,000, or 30,000 to 50,000individuals.

A panel of iPSC lines may include iPSC lines from at least two ethnicgroups, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or 50 ethnicgroups. Examples of ethnic groups include, but are not limited to,Europeans, Japanese, Chinese, and the Yoruba of Nigeria, and ethnicgroups listed in Table 4.

TABLE 4 Exemplary Ethnic Groups Africa Bantu Biaka Mandenka Mbuti pygmyMozabite San Yoruba Native America Colombian Karitiana Maya Pima SuruiAsia Ctrl/South Balochi Brahui Burusho Hazara Kalash Makrani PathanSindhi Uyghur Western Asia Bedouin Druze Eastern Asia Cambodian Dai DaurHan (N. China) Han (S. China) Hezhen Japanese Lahu Miao Mongola NaxiOroqen She Tu Tujia Xibo Yakut Yi Europe Adygei Basque French NorthItalian Orcadian Russian Sardinian Tuscan Oceania Melanesian Papuan

IV. Methods for Use of Induced Stem Cell Lines and Panels of InducedStem Cell Lines

A. Overview

The iPSC lines and panels of iPSC lines described herein are useful in anumber of methods relating to drug discovery and development. Typically,a drug candidate compound will be evaluated in a biochemical assay(e.g., a receptor binding assay) that evaluates only a single or veryfew sequence variants of the drug target expressed in a patientpopulation. Thus, such assays provide little information as to howeffective the drug candidate compound is likely to be in patients thatexpress a drug target allele that differs from the particular drugtarget allele that was originally screened. Along the same lines, drugcandidate compounds often undergo functional cellular screens in one orfew cell lines engineered to express a specific allele of the drugtarget, again ignoring the genetic diversity of a human patientpopulation not only with respect to the drug target itself, but also tothat of the various downstream signal transduction proteins that play arole in the response endpoint of cells to a drug. Likewise, adverseeffects of candidate drug compounds (e.g., liver toxicity) are generallyevaluated in inbred animal models, which are likely to be uninformativefor a variable fraction of a human patient population. In contrast, drugscreening in panels of genetically diverse iPSC lines, as describedherein, addresses the lack of genetic diversity in the prevailing drugscreening models.

The panels of genetically diverse iPSC lines described herein (e.g.,human iPSC lines) or cells differentiated from panels of geneticallydiverse iPSC lines, as described herein, may be used to identify testcompounds that act on a drug target of interest. In some embodiments,the panels of iPSCs cell lines include a sufficient number of iPSC linessuch that at least two, e.g., at least 3, 5, 10, 20, 50, 100, or 200polymorphic alleles of a drug target (e.g., a GPCR, ion channel, orkinase) are represented in the panel. In some embodiments, panels ofiPSC lines are derived from subjects diagnosed as suffering from ahealth condition or identified as having a predisposition to the healthcondition. In other embodiments, the iPSC line panels comprise iPSClines each of which that has at least one polymorphic allele associatedwith a health condition or a predisposition to the health condition.

Drug targets for many health conditions are known. Such drug targets mayinclude, but are not limited to, receptors, GPCRs, growth factorreceptors, neurotransmitter receptors, ion channels, enzymes, proteinkinases, proteases, cytoskeletal proteins, and transcription factors.Test compounds can be assayed for their effect on a drug target by anumber of assays known in the art. Such assays include cell-based assaysincluding, but not limited to, assays for determining second messengerlevels, e.g., intracellular calcium, cAMP, cGMP, arachidonic acid, andinositol phosphates; channel currents; apoptosis; proliferation;morphological changes; changes in adhesion. Examples of cell-basedassays include, but are not limited to those described in, U.S. Pat.Nos. 7,319,009, 7,288,368, and 7,238,213, Cell based assays may alsoinclude determining the cellular localization of one or more proteins(e.g., protein kinases, receptors, and transcription factors) in cellsin the presence or absence of a test compound. Test compounds may alsobe screened for their ability to alter a gene expression profile by anygene expression profiling method known in the art. In some cases, thecells to be screened may be genetically modified to express one or morereporter proteins that can indicate activation of a signaling pathway.For example protein-protein interactions between fusion proteinsintroduced into cells may be detected by a number of methods known inthe art, e.g., by fluorescence resonance energy transfer (FRET) orenzyme fragment complementation.

In some cases, the mechanistic basis of a sporadic form of a disease isa combination of genetically-determined cell type-specific phenotype andepigenetic factors (e.g., oxidative stress). In other words,iPSC-derived differentiated cells from a patient with a sporadic form ofa disease (e.g., Parkinson's) may bear a genetic predisposition to apathological or pre-pathological cellular phenotype (e.g., apoptosis),but the phenotype may only manifest in vitro in the presence of anappropriate “stressor” that recapitulates environmental epigeneticfactors associated with the sporadic disease or a cellular phenotypesthat precede a clinical manifestation of the disease (e.g.,mitochondrial dysfunction, oxdidative stress, or nitrosylative stress).Accordingly, in some cases disease-relevant cellular phenotypes areinduced by a stressor. Examples of stressors include, but are notlimited to cellular oxidative stress, nitrosylative stress, proteasomeinhibition, inhibition of mitochondrial electron transport, translationinhibition, decreased calcium buffering, high osmolarity, heat shock,heavy metals (e.g., Zn, Mn, Fe, Cd, Al, or Pb), protein misfolding.Examples of agents that induce, increase, or result from oxidativestress include, but are not limited to, H₂O₂, ascorbic acid/FeSO₄,4-hydroxynonenal, glutamate, kainate, NMDA, dopamine, okadaic acid,Aβ¹⁻⁴² and isocyanate. Proteasome inhibitors include, but are notlimited to lactacystin, ziram, MG 132, andcarbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal(PSI). Mitochondrial stressors include, but are not limited to,rotenone, 3-nitropropionic acid (NPA), 1-methyl-4-phenylpyridinium(MPP+), antimycin, paraquat, methylglycoxal, and cyanide. Nitrosylativestressors include, but are not limited to,(±)-S-nitroso-N-acetylpenicillamine, sodium nitroprussiate, andperoxynitrite.

In some cases, the stressor is provided by expressing or overexpressingan exogenous wild type or mutated gene and/or protein. Examples of suchgenes include, α-synuclein, amyloid beta, Aβ¹⁻⁴², Parkin, Pink1,Leucine-rich repeat kinase 2 (LRRK2), superoxide dismutase (SOD).

Assays of drug candidate compounds in an iPSC line or a panel of iPSClines can include determining a dose-response. In some embodiments, thedose response of an iPSC line or that of one or more types of cellsdifferentiated from the iPSC line provides an indication that of thelikely efficacy of the compound in the corresponding iPSC donor. In someembodiments, the fraction of iPSC lines in a panel of iPSC lines thatexhibit an acceptable dose-response to a test compound indicates anexpected probability of an acceptable dose-response relationship in thetarget patient population of interest. In some cases, cell-based assaysof drug candidate include a comparison of responses obtained in a panelof iPSC lines or iPSC-derived cells to one or more reference iPSC linesor cells that serve as a positive or negative control for the effect ofa drug candidate compound. The reference iPSC lines or cells may be froma healthy iPSC donor, from an iPSC donor diagnosed as suffering from ahealth condition, or an iPSC donor carrying a polymorphic alleleassociated with a health condition. In other embodiments, assays of drugcandidate compounds in an iPSC line or a panel of iPSC lines can includedetermining effective concentrations, maximum tolerated dose and minimumeffective concentration. Additional methods and assays are disclosed inU.S. application No. 61/061,594; filed Jun. 13, 2008; First InventorKazuhiro Sakurada, hereby incorporated by reference.

In some cases, the drug screening may be conducted on cellsdifferentiated from iPSCs. Examples of such differentiated cells aredescribed herein (e.g., hepatic cells, neural stem cells, neurons,pancreatic beta cells, cardiomyocytes, hepatic stem cells,oligodendrocytes). The drugs may be targeted to treat a specific diseaseor condition, e.g., a disease or condition described herein. Forexample, the iPSCs may be differentiated into dopaminergic neurons,which are used to screen drugs for Parkinson's disease. In other cases,neurons or neural stem cells differentiated from iPSCs may be used toscreen drugs for treating Alzheimer's disease, multiple sclerosis, orother neurological disorders. In some cases the In other cases, theiPSCs may be transplanted directly into an immunocompromised animal,e.g., SCID mouse, which is then used to establish in vitro or in vivoassay systems that mimic physiologic conditions in humans or otheranimals. The in vitro or in vivo assay systems may be used to screen fordrugs, e.g., drugs for Parkinson's disease, or as a means to identifybiological mechanisms.

Screening of test compounds may also be conducted in iPSC-derived cellswhen an abnormal cellular phenotype (e.g., abnormal cell morphology,gene expression, or signaling), associated with a health condition or apredisposition to the health condition is known, but a drug target hasnot yet been identified. Such assays may include contacting a testpopulation of iPSC-derived cells from one or more iPSC donors with atest compound and contacting with a negative control compound a negativecontrol population of iPSC-derived cells from the same one or more iPSCdonors. The assayed cellular phenotype associated with the healthcondition of interest in the test and negative control populations canthen be compared to a normal cellular phenotype. Where the assayedcellular phenotype in the test population is determined as being closerto a normal cellular phenotype than that exhibited by the negativecontrol population, the drug candidate compound is identified asnormalizing the phenotype. A normal cellular phenotype with respect to aparticular health condition or a predisposition for a health conditionmay be established in iPSC-derived cells from iPSC donors that do notsuffer from the health condition or a predisposition for the healthcondition.

Test compounds identified as lead compounds, may be tested on a panel ofiPSC-derived cells in a manner analogous to a clinical trial. In somecases, the efficacy of the lead compound versus a negative controlcompound, e.g., a placebo compound is determined in a panel ofiPSC-derived cells from patients suffering from the same healthcondition. Preferably, such a panel of iPSC-derived cells is fromsubjects that are genetically diverse. For example, such patients may becarry at least one polymorphic allele that is unique among theiPSC-derived cells to be included in the panel, e.g., 5 to 10, 20 to 50,50 to 200, 200 to 500, 500 to 1000, 1000 to 5000, 5000 to 20000, or20000 to 50000 polymorphic alleles that are unique within the panel ofiPSC lines. A number of methods for quantifying the genetic diversity ofa population are known in the art, e.g., the analysis of molecularvariance (AMOVA) and generalized analysis of molecular variance(GAMOVA). See, e.g., Excoffier et al (1992), Genetics, 131: 479-491;Nievergelt et al (2008), PLOS Genetics, 3(4):e51. Various clinicalexperimental designs known in the art may be used for comparing theeffect of a lead compound versus a negative control compound. See, e.g.,Chow et al (2004) “Design and Analysis of Clinical Trials: Concepts andMethodologies,” John Wiley & Sons, Inc., Hoboken, N.J.

The efficacy of the lead compound in iPSC-derived cells may bedetermined based on any cellular response endpoint, e.g., a responseobtained in any of the cell-based assays or gene expression profilingassays mentioned herein.

In some cases, potential adverse effects of a lead compound are testedon a panel of iPSC-derived cells. The iPSC-derived cells may include anycell type that hepatocytes, cardiomyocytes, neurons,

Drug candidate compounds may be individual small molecules of choice(e.g., a lead compound from a previous drug screen) or in some cases,the drug candidate compounds to be screened come from a combinatoriallibrary, i.e., a collection of diverse chemical compounds generated byeither chemical synthesis or biological synthesis by combining a numberof chemical “building blocks.” For example, a linear combinatorialchemical library such as a polypeptide library is formed by combining aset of chemical building blocks called amino acids in every possible wayfor a given compound length (i.e., the number of amino acids in apolypeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks. Indeed,theoretically, the systematic, combinatorial mixing of 100interchangeable chemical building blocks results in the synthesis of 100million tetrameric compounds or 10 billion pentameric compounds. See,e.g., Gallop et al. (1994), J. Med. Chem 37(9), 1233. Preparation andscreening of combinatorial chemical libraries are well known in the art.Combinatorial chemical libraries include, but are not limited to:diversomers such as hydantoins, benzodiazepines, and dipeptides, asdescribed in, e.g., Hobbs et al. (1993), Proc. Natl Acad. Sci. U.S.A.90, 6909; analogous organic syntheses of small compound libraries, asdescribed in Chen et al. (1994), J. Amer. Chem. Soc., 116: 2661;Oligocarbamates, as described in Cho, et al. (1993), Science 261, 1303;peptidyl phosphonates, as described in Campbell et al. (1994), J. Org.Chem., 59: 658; and small organic molecule libraries containing, e.g.,thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974),pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134), benzodiazepines(U.S. Pat. No. 5,288,514).

Numerous combinatorial libraries are commercially available from, e.g.,ComGenex (Princeton, N.J.); Asinex (Moscow, Russia); Tripos, Inc. (St.Louis, Mo.); ChemStar, Ltd. (Moscow, Russia); 3D Pharmaceuticals (Exton,Pa.); and Martek Biosciences (Columbia, Md.).

B. Individualized Drug Therapy and Failed Drug “Rescue”

iPSC cell lines and iPSC-derived cells generated from a subject (e.g., ahuman subject) can be used to determine the likelihood that a particulardrug will have sufficient efficacy in that subject and, if so, anappropriate dose range for that subject. This process is illustratedschematically in FIG. 3. iPSC-derived cells from a subject, e.g.,differentiated iPSC-derived cells may be exposed ex vivo to a drug to betested, and then assayed for their phenotypic response to the drug asdescribed herein. The response of the iPSC-derived cells may be comparedto a reference response obtained in iPSC-derived cells from one or moreindividuals in which the drug has been shown to be effective and/or areference response in iPSC-derived cells from subjects in which the drugwas found to be ineffective. In some cases, the subject to be tested isa subject suffering from a health condition or a predisposition to thehealth condition. For example, where the subject is suffering from ahealth condition, and multiple drugs are available to treat the healthcondition, the efficacies and adverse effects of the multiple drugs maybe evaluated iPSC-derived cells from that individual. Preferably, theiPSC-derived cells used to test drug efficacy include cells that expressat least one drug target (e.g., a neurotransmitter receptor). In othercases, the subject is not suffering from a health condition. In oneembodiment, drugs for various health conditions are tested preemptivelyin iPSC-derived cells from a healthy subject to establish apharmaco-phenomic profile for that subject. The pharmaco-phenomicprofile may subsequently be used as needed for selecting optimal drugsand drug dosing for treatment of the particular subject.

C. Disease Pathway and Target Discovery

For many diseases, especially those that have primarily a sporadic form(e.g., Parkinson's disease), the underlying cellular phenotype(s) thatprecede and eventually result in pathology are unknown. In fact, forprogressive degenerative conditions, it is likely that a causative orpredictive cellular phenotype occurs well before the first manifestationof symptoms. However, for many types of diseases the relevant cells(e.g., neurons, cardiomyocytes, and pancreatic cells) are not directlyaccessible for analysis. Thus, depending on the cell type affected by aparticular disease, it has not been possible to compare live cells frompatients to those of normal subjects in order to identifydisease-relevant, cellular phenotypes that cause or predispose for adisease. Identification of reproducible cellular phenotype differencesbetween patient iPSC-derived and normal subject iPSC-derived cellsallows the development of screening assays to identify candidatetherapeutic agents. Candidate therapeutic agents are those thatnormalize a disease-associated cellular phenotype, i.e., alter therelevant cellular phenotype in the patient-derived cells so that it iscloser to the corresponding cellular phenotype in cells derived fromnormal subjects under the same conditions. Alternatively, thetherapeutic agent may alter a cellular phenotype of patient-derivediPSCs so as to protect them from a stressor, as described herein.

Sets of data representing various cellular phenotypes (e.g.,mitochondrial ROS production, expression profiles, protein aggregation)in patient iPSC-derived cells versus normal subject iPSC-derived cellsconstitute vectors in a multidimensional space, amenable to analysis bymeans of multivariate and univariate statistical and machine learningtechniques. Thus cellular phenotypes distinguishing patient versusnormal subject can be identified, for example, by means of univariatestatistical methods, such as t-test, ANOVA, regression, as well as theirnon-parametric analogs. In some embodiments, cellular phenotype data arefurther filtered using various statistical criteria, e.g., p-value ofsignificance (Type1 error), effect size, etc. Sets of cellularphenotypes which differ significantly between disease and normal statesare further scrutinized by biological pathways analysis. In many cases,a pathway enrichment analysis is performed to further narrow the set ofcellular phenotypes which are the most disease-informative. A number ofstatistical procedures such as Hypergeometric statistic,Kolmogorov-Smirnoff test, etc, can be used to perform pathway enrichmentanalysis.

In some cases, cellular phenotypes that are found to differsignificantly in patient versus normal subjects (i.e., disease-relevantcellular phenotypes) need to be validated by means of orthogonal assays.In other cases, the identified disease-relevant cellular phenotypes areconfirmed by performing validation/cross-validation analysis on theindependent data sets from the same type of cellular phenotype assays.In some embodiments, disease-relevant cellular phenotypes are determinedby first assaying and analyzing only a portion of the available patientand normal iPSC lines, and then validating disease-relevant cellularphenotypes in the remaining iPSC lines. In other embodiments, where itis not feasible to utilize independent sets of iPSC lines fordisease-relevant cellular phenotype discovery versus validation, otherstatistical approaches, such as k-fold cross-validation techniques, areused instead. In some cases, one or more validated cellular phenotypesis then used to assess test agents for their ability to convert one ormore cellular phenotypes reflecting a disease condition to cellularphenotypes reflecting a normal condition.

Where the disease under study is a progressive condition with apotentially late onset (e.g., age 60 and over), selection of “normal”control subjects is non-trivial, as it is usually not possible to know,prospectively, who will develop a progressive degenerative disorder. Inother words, subjects that are apparently normal at a given age/timepoint (e.g., when a biopsy is obtained for iPSC derivation) mayeventually develop the disease for which an associated cellularphenotype is sought. Thus, cells derived from such a subject would notbe a valid “normal” control. Accordingly, in some embodiments, ratherthan selecting an age-matched normal control subject, a “wellderly”subject is selected for normal control iPSC derivation. As used herein,a “wellderly” subject refers to any subject that is at least 80 yearsold and has not suffered from any major chronic diseases. Selection ofwellderly individuals as normal control subjects makes it statisticallyless likely that such individuals will go on to develop a degenerativecondition. Thus, iPSCs derived from such individuals are less likely toexhibit a cellular phenotype that is associated or predictive of thedisease being analyzed, and therefore provide a more reliable “normalcontrol” phenotype for purposes of comparison to patient-derived iPSCsand iPSC-derived cells. In other embodiments, elderly individuals areselected for control iPSC generation that while not having suffered froma degenerative disease under study, may have suffered other unrelateddegenerative diseases. In other cases, age-matched subjects free of thedisease to be analyzed are used to generate normal control subjectiPSCs.

In some cases, once a candidate therapeutic agent has been identified aseffectively normalizing a cellular phenotype in a small number ofpatient iPSC lines and cells derived therefrom, efficacy is tested inlarger panels of patient iPSC-derived cells to identify potentialvariation in efficacy or toxicity of the candidate therapeutic agent. Insome cases, efficacy is tested in iPSCs or iPSC-derived cells from atleast about 20 to about 500 patients, e.g., at least about 25, 30, 40,50, 60, 70, 100, 200, 250, 300, 400, or another number of patients fromat least about 20 to about 500 patients. In some embodiments, biomarkersassociated with responsiveness to a candidate therapeutic agent or lackof responsiveness to a candidate therapeutic agent are identified andused to stratify a patient population into, e.g., “high responders” (HR)and “low responders” (LR), as schematized in FIG. 4. In some cases,biomarkers are used to identify suitable patients for clinical trials ofa candidate therapeutic agent. In other cases, biomarkers are used topredict the responsiveness or potential toxicity of a therapeutic agentfor particular patients. In some cases, biomarkers include genomicbiomarkers (e.g., SNPs, a CNVs, or other genetic polymorphisms). Inother cases, the biomarkers include an expression profile signature(e.g., an mRNA expression profile). The biomarkers may include a proteinexpression profile or even a single protein expression level. In somecases, where the biomarkers are expression profile biomarkers, these maybe determined directly from a patient sample (e.g., blood, urine,sputum, hair, skin, or other biological sample taken directly from thepatient). In other embodiments, expression profile biomarkers, arespecific to patient iPSCs or iPSC-derived cells in which the candidatetherapeutic agent or therapeutic agent is tested.

Thus, in some embodiments, iPSCs are derived from patients and controlsubjects, and the iPSCs are differentiated into disease-relevant celltypes thereby allowing a comparison of cellular phenotypes inpatient-derived cells versus normal subject derived cells. For example,the cellular phenotype that is compared may include a mitochondrialphenotype, e.g., ATP synthesis, ATP/ADP ratio, mitochondrial potential,calcium buffering, production of reactive oxygen species, mitochondrialfusion and fission, mitochondrial morphology, and mitochondrialmovement. In other cases, the cellular phenotype that is compared is thefraction and rate at which a particular cell type is undergoingapoptosis in the presence of a stressor. In some embodiments, thecellular phenotype is protein aggregation (e.g., the formation of lewybodies). In other embodiments, gene expression (e.g., microRNAexpression) is compared between patient-derived cells and normalsubjects.

In some cases, relational databases are constructed that integratemultiple data streams relating to each patient and control iPSC line.These data include, but are not limited to, one or more of thefollowing: patient medical history and family medical history, patientmedical data (e.g., blood pressure, liver enzyme levels), patientadverse drug reactions, patient drug responsiveness, partial or completegenomic sequence, sequence of all genes with known disease-associatedalleles, comprehensive SNP genotypes (e.g., genotypes for all SNPs withknown disease associations), gene copy number variation (CNV)polymorphisms, expression profiles for iPSCs and for cellsdifferentiated from the iPSCs (e.g., dopaminergic neurons, corticalneurons, motor neurons, pancreatic cells, hepatocytes, cardiomyocytes,and vascular epithelial cells) under resting and under various stimulusparadigms (e.g., in the presence of a stressor), all cellular phenotypeassay data used for initial pathway discovery and for drug screening,including, e.g., cellular phenotype data in the presence or absence oftest compounds and compounds with known pharmacological properties(e.g., a cholinesterase inhibitor, a receptor ligand, a kinase inhibitoretc.). In some embodiments, a user can query such a database based onany set of criteria with user define limits. For example, a user maywish to identify polymorphisms associated with patients whoseiPSC-derived dopaminergic neurons did or did not respond to a candidatetherapeutic agent. In another example, a user may wish to identify acommon gene expression profile that distinguishes motor neurons thatshowed a severe apoptotic response to a stressor versus a mild apoptoticresponse, etc. Such databases are very useful for data mining andestablishing robustly predictive signatures for specific disease statesand their response to candidate therapeutic agents.

EXAMPLES Example 1 Generation of iPSC Lines from Patients Suffering fromSpinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is a neuromuscular disease characterizedby degeneration of motor neurons that is among the leading causes ofchildhood paralysis and mortality. The disease exhibits a wide range ofseverity affecting infants through adults, and is subdivided into typesI-IV based on the age of onset and severity of symptoms: Type I“Infantile” onset at ages 0-6 months and generally fatal); Type II“Intermediate,” onset at ages 7-15 months; inability to stand or walk,but some ability to maintain a sitting position; Type III “Juvenile”onset at ages 18 months to 17 years, with some ability to walk, thoughpotentially transient; Type IV “Adult,” some muscle weakness, but nogenetic basis is known.

The molecular basis of SMA is linked to the Survival Motor Neuron (SMN)gene. The region of chromosome 5 that contains the SMN (survival motorneuron) gene has a large duplication. A large sequence that containsseveral genes occurs twice—i.e. once in each of the adjacent segments.The two copies of the gene—known as SMN1 and SMN2—differ by only a fewbase pairs. The SMN2 gene contains a mutation that occurs at the splicejunction of intron 6 to exon 7 resulting in about 90% of SMN2 pre-mRNAtranscripts being spliced into a form that excludes exon 7. This shortermRNA transcript codes for a truncated SMN protein, which is rapidlydegraded. About 10% of pre-mRNA transcript from SMN2 is spliced into thefull length transcript that codes for the fully functional SMN protein.This splicing defect occurs in multiple cell types, although, forunknown reasons, the survival of motor neurons appear to be particularlyaffected.

SMA results from the loss of the SMN1 gene from both chromosomes, andits severity, ranging from SMA 1 to SMA 3, largely depends on whetherthe level of SMN2^(E7) transcript can make up for low levels or absenceof exon 7-inclusive SMN 1 transcript. The mutations that cause the lossof SMN 1 are of two types. Deletion mutations, in which both copies ofthe SMN1 are missing. The other type of mutation is a conversionmutation in which both copies of the SMN1 gene have a point mutationresulting in the same splicing pattern as the SMN2 gene. As an initialstep towards developing an in vitro assay for identifying molecules thatcan increase levels of exon 7-inclusive SMN2 (SMN2^(E7)) transcript, wegenerated several iPSC lines from Coriell fibroblast lines establishedfrom three SMN1^(−/−) SMA patients and from two healthy SMN1^(−/+)subjects.

Induction of iPSCs was initiated by transduction of SMN1^(−/−) andSMN1^(−/+) fibroblast cultures with four MoMLV VSV-G-pseudotyped virusesfor expression of human OCT4, SOX2, KLF4, and c-MYC, each at an MOI ofabout 10. Five days after viral transduction, fibroblasts were switchedfrom human fibroblast medium into human ES cell supportive medium andmonitored daily for the appearance of putative iPSC colonies based onmorphological criteria.

Initial putative SMA-iPSC colonies were picked after approximately threeweeks and propagated clonally in the presence the presence of the ROCKinhibitor Y-27632 (10 μM) Calbiochem) to derive the SMN1^(−/−) iPSClines SM4p, SM7t, and SM8c, and the SMN1^(−/+) iPSC lines SM9a andSM10d, as shown in FIG. 5. Each of the iPSCs expressed the pluripotencyassociated markers, Nanog, Oct4, SSEA3, SSEA4, TRA1-60, and TRA1-81(data not shown) as determined by immunocytochemistry. Q-PCR analysisshowed that these iPSC lines expressed endogenous Oct 4, Sox2, and Klf4,but not the exogenous Oct4, Sox2, and Klf4 introduced by viraltransduction. In addition, Q-PCR analysis also demonstrated expressionof Nanog, SSEA-3, SSEA-4, TRA1-60, TRA1-81, DNMT3B, FOXD3, LIN28,ZNF206, LEFT2, TDGF1, and TDGF2 in all of the iPSC lines (data notshown). Importantly, all of the SMA iPSC lines were able to formembryoid bodies (EBs) as shown in FIG. 6, which indicated that theselines had good potential for differentiation as is expected for iPSCs.Indeed, the ability of the SM8c line to differentiate into ectodermal,mesodermal, and endodermal lineages in vitro was confirmed byimmunostaining for the ectodermal marker TuJ1, the mesodermal markerDesmin, and the endodermal marker AFP, as shown in FIG. 7. Further, theSM8c iPSC line was shown to differentiate into mature motor neurons asshown by double immunolabeling for Islet and Neuro-N (data not shown).

Based on these results, we concluded that iPSCs can be generated fromSMA patients and differentiated into motor neurons, as required for thescreening assay described in Example 2.

Example 2 Assay for Identification of Molecules that Improve Molecularand Cellular Disease Phenotypes in Motor Neurons from Patients Sufferingfrom Spinal Muscular Atrophy

We seek to identify molecules that increase the level of SMN2^(E7)transcript in motor neurons derived from patients suffering from SMA. Inprinciple, increased levels of SMN2^(E7) transcript can be increased byboosting SMN2 transcription, reducing degradation of SMN2 mRNA, or byincreasing the fraction of SMN2 pre-mRNA that is spliced into SMN2^(E7)mRNA. SMA patient-specific motor neurons are obtained by firstgenerating panels of iPS cell lines from Type I, Type II, and Type IIISMA patients, as described in Example 1, and subsequentlydifferentiating iPSCs into motor neurons. Prior to motor neurondifferentiation SMA patient SMN2 minigene reporter iPSC lines areestablished to provide a convenient readout for the level of SMN2^(E7)transcript in motor neurons.

Following parental informed consent, standard dermal punch biopsies 2-4mm in diameter and thickness are obtained from approximately 30 Type I,30 Type II, and 30 Type III SMA patients, all of whom have an SMA1^(−/−)genotype, and 10 healthy, age-matched control subjects that have anSMA1^(−/+) genotype. For each SMA-iPSC line to be generated, thefollowing corresponding patient information is collected and annotatedin an iPSC line database: disease severity ranking (i.e., Type I, II, orIII), age of disease onset, patient medical history, family medicalhistory including incidence of ALS, blood level of SMN protein, SMN1 andSMN2 genotypes, MUNE Motor Unit Number Estimation, Hammersmith SMAFunctional Motor Scale ranking, breathing test evaluation (only forchildren>5 yrs), symptom progression evaluation (e.g., how outcome ofmotor tests has changed over time), muscle mass index, description oftherapeutic interventions to date, and therapy response. Additional datamay be added to each record as they are acquired, including, e.g., SMNprotein levels and SMN2^(E7) transcript levels under variousexperimental conditions (e.g., in the presence or absence of a candidatetherapeutic compound), informative SNP genotypes, genomic sequence, andtissue/cell-type specific expression profiles.

Biopsy samples are stored for up to 5-7 days at 4° C. in a “biopsymedium” containing KO-DMEM and supplemented with 10% fetal bovine serum(FBS), Earl's Salts, nucleosides, beta-mercaptoethanol (BME),non-essential amino acids, glutamine, and penicillin/streptomycin.Biopsies are minced into 4-5 pieces, and the pieces are then transferredto a 60 mm dish. The pieces are then “sandwiched” under an acid-washedcoverslip and cultured in biopsy medium for five days. Subsequently, thesandwiched biopsy explants are cultured in human fibroblast (“hFib”)medium containing KO-DMEM, Earl's Salts, 10% FBS, glutamine,penicillin/streptomycin, and medium is replaced every 3-4 days until thecoverslip is confluent. SMA iPSCs are generated, as described in Example1, from fibroblasts obtained from each biopsy.

An SMN2 splicing minigene reporter construct is generated thatincorporates exons 6, 7, and 8, and utilizing the SMN2 promoter isgenerated essentially as described in Zhang et al (2001), Gene Ther.,8:1532-1538 and Wilson et al, Stem Cells and Development, 16:1027-1041.The SMN2 reporter construct will incorporate the DD-AmCyan1 fluorescentprotein reporter to maximize the signal to noise ratio in a compoundscreening reporter gene assay. The DD-AmCyan1 protein contains adegradation (“DD”) domain that conditionally destabilizes the proteinthereby keeping “background” levels of the reporter protein prior to atest compound screening assay very low. However, upon addition of thecell-permeable “Shield1” ligand (Invitrogen), which selectively binds tothe DD domain, the reporter protein is stabilized and can thereforeaccumulate. Thus, potential differences in DD-AmCyan1 reporter levels inthe presence or absence of test compounds are maximized by measuringalmost exclusively reporter protein produced after the beginning of thescreening assay, i.e., after the addition of the Shield1 ligand and testcompound. Additional reporter constructs will include AmCyan1 orluciferase as the reporter. Other constructs will include the CMVpromoter to drive SMN2 minigene expression. The SMN2 reporter constructis then stably transfected into type I, type II, and type III SMA-iPSCsand healthy control (SMN1^(WT/WT)) iPSCs to generate SMN2-reporterSMA-iPSC lines of varying disease severity backgrounds, and SMN2reporter control iPSC lines, respectively. Primary screening of testcompounds for the ability to increase properly spliced SMN2 transcriptlevels is conducted initially in motor neurons derived from Type I SMAreporter iPSCs.

On day 0, confluent 10 cm plates of SMN2 reporter SMA-iPSCs aretrypsinized and then washed/resuspended in embryoid body (EB) mediumcontaining KO DMEM (Invitrogen, catalog #10829-018), Knockout SerumReplacement (Invitrogen, catalog #A1099202), Plasmanate (Talecris),Glutamax (Invitrogen, catalog #35050079), non-essential amino acids(Invitrogen, cat #11140050). After washing and resuspension, the cellsare plated in ultra-low attachment (ULF) 6-well plates and grown intoEBs over the next 4-5 days. On day 5, EBs are washed, gently resuspendedin EB medium, and replated in a new ULA 6-well plate, and thewash/replate procedure is repeated on day 8 or 9. On day 11, EBs arecollected and resuspended in N2 base medium (DMEM/F12, Glutamax(Invitrogen, catalog #10565), N-2 Supplement (Invitrogen, catalog#17502-048), D-Glucose (Sigma, catalog #G8769), Ascorbic Acid (Sigma,catalog #A4403-100 mG)) supplemented with 1 μM Retinoic Acid (RA) and100 nM Purmorphamine. (PM). On day 14, EBs are transferred to in N2 Basemedium+1 μM RA+1 μM Purmorphamine and replated (3 ml of EBsuspension/well) on ULA 6 well plates. N2 base

The RA (1 μM)/PM (1 μM)-supplemented EB medium is replaced every 3-5days, as needed, until approximately day 28. Afterwards, EBs aredissociated by dilute papain treatment and gentle trituration, and thenreplated on new ULF 6-well plates followed by gentle trituration every10 minutes over a period of 45 minutes. After dissociation, theresulting cell suspension is collected and transferred to a 50 mlconical tube containing motor neuron maturation medium (DMEM/F12,Glutamax, N-2 Supplement (Invitrogen, catalog #17502-048), B-27Supplement (Invitrogen, catalog #17504-044), D-Glucose, Ascorbic Acid(Sigma, catalog #A4403-100 mG), 2 ng/mL each GDNF (R&D, catalog#212-GD), BDNF (R&D, catalog #248-BD), and CNTF (R&D, catalog#257-NT/CF). The cell suspension is pelleted by centrifugation at 1000RPM for five minutes, and is then resuspended in motor neuron maturationmedium at a cell concentration of approximately 1.6×10⁶ cells/ml.Aliquots (50 μl) of cell suspension are then plated on laminin-coatedwells of optical grade 96 well plates. Beginning on day 31, half-mediumchanges are conducted every other day or every day depending on howquickly the medium becomes spent. The differentiated cultures aremaintained in motor neuron differentiation medium for another four weeksprior to beginning SMN2-reporter assays to allow expansion andmaturation of the motor neuron population.

At the beginning of the screening assay, all wells of 96-well platemature motor neuron (MMN) cultures are incubated in the presence ofShield1 is at a final concentration of 1 μM. Test wells are incubated inthe presence of test compounds from the NIH Clinical Collection Library(available from BioFocus DPI) at a final concentration of 50 μM.Negative control wells receive no addition or are incubated with avehicle compound (e.g., DMSO) at a concentration equivalent to thatpresent in some of the test compound solutions. Positive control wellsare incubated in the presence of sodium vanadate (50 μM), which haspreviously been shown to significantly increase levels of SMN2^(E7)transcript (Zhang et al (2001), Gene Therapy, 8, 1532-1538). Afterincubation for 24 hours, cultures are fixed and processed forimmunofluorescence detection of Islet 1/2 (mature motor neurons) andOlig2 (motor neuron progenitors) and DD-AmCyan1 fluorescence levels areimaged and quantified in Islet 1/2⁺ and Olig2⁺ cells. Compounds thatincrease SMN2 reporter levels (“candidate therapeutic” compounds) arescreened in secondary assays for their ability to increase SMN2^(E7)transcript levels and for their ability to promote SMA motor neuronsurvival over a period of about two weeks. Candidate therapeuticcompounds are then tested on motor neurons derived from additional typeI SMA SMN2-reporter iPSC lines, and from type II and type III iPSC linesto validate the effect of the therapeutic candidate compounds on motorneurons from diverse genetic backgrounds extant in the SMA patientpopulation.

It is expected that identification of compounds that increase the netlevel SMN2^(E7) transcript in patient-derived motor neurons is likely tobe more relevant for identification of therapeutic drug candidates forSMA than a similar assay in cell types relatively unaffected or lessaffected by loss of SMN1 (e.g., fibroblasts) or heterologous cell lines.

Example 3 Generation of iPSC Lines iPSCs from Patients with IdiopathicParkinson's Disease and Defined Mutations in Genes Associated withParkinson's Disease

Parkinson's Disease (PD) is one of the most common neurodegenerativediseases of aging, affecting 1-2% of the population over 65 years ofage. Clinical symptoms include rest tremor, bradykinesia, and rigidity.We seek to generate a PD patient iPSC model to identify candidatetherapeutic agents that slow, halt, or reverse PD progression.

iPSC lines are generated from skin biopsies obtained from 10 healthycontrol subjects with no known family history of PD, 10 patients withsporadic PD, patients each with mutations in the genes that encodeα-synuclein (PARK1), parkin (PARK2), PINK 1 (PARK6), or LRRK2 (PARK8)for a total of 10 patients for each mutation. iPSCs are generated asdescribed in Example 1. Afterwards, dopaminergic neurons are derived bydifferentiating each of the patient iPSC lines and control subjectiPSCs. A dopaminergic phenotype is established by immunocytochemicalstaining for tyrosine hydroxylase positivity, and assaying thedifferentiated cells for the ability to synthesize and release dopamine.Dopaminergic neurons are obtained by differentiating the iPSCs accordingto the method of Perrier et al (2004), Proc Natl Acad Sci USA 101,12543-12548. After validating the dopaminergic phenotype of neuronsdifferentiated from each of the above iPSC lines, cultures of thepatient iPSC-derived dopaminergic neurons are tested in a battery ofcellular phenotype assays and compared to control subject dopaminergicneurons. These are: assays for aggregation of α-synuclein, dopaminergicneuron apoptosis (TUNEL, caspase activation) and necrosis (CytoTox-Glo),oxidative stress indicators (glutathione levels, ROS, and 4-HNE), andmitochondrial dysfunction (ATP content, membrane potential, morphology,and calcium buffering). It is expected that sporadic forms of PD and PDcaused by the above-mentioned mutations will exhibit very similardopaminergic cellular phenotypes in at least some of these assays. Oncethis is established, one of more of the PD-associated cellularphenotypes is used as the basis of a screen for candidate therapeuticagents that can reverse or ameliorate these cellular phenotypes.Further, it is expected that as the PD cellular phenotypes areidentified in disease relevant cells (dopaminergic neurons) from humanPD patients, their predictive value and reliability for the developmentof therapeutic agents will be more robust than those based onheterologous assay models.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for identifying an agent that corrects a phenotypeassociated with a health condition or a predisposition for the healthcondition, comprising: (i) contacting a first population of isolatedcells differentiated from a human induced pluripotent stem cell line,wherein said cells exhibit a phenotype associated with the healthcondition or predisposition for the health condition, with a candidateagent; (ii) contacting a second population of isolated cellsdifferentiated from the human induced pluripotent stem cell line,wherein said cells exhibit a phenotype associated with the healthcondition or predisposition for the health condition, with a negativecontrol agent; (iii) assaying a phenotype in the first population andsecond population after the contacting steps; and (iv) identifying thecandidate agent as correcting the phenotype if the assayed phenotype ofthe first population after the contacting step is closer to a normalphenotype than the phenotype of the second population after thecontacting step; wherein one or more cells differentiated from theinduced pluripotent stem cell line comprise an exogenous Oct3/4 gene, anexogenous Sox2 gene, and an exogenous Klf4 gene; wherein the cells inthe first and second populations of human induced pluripotent stemcells: (a) comprise at least one endogenous allele associated with thehealth condition or the predisposition for the health condition; or (b)are generated from a subject suffering from the health condition or thepredisposition for the health condition.
 2. The method of claim 1,wherein the health condition is a neurodegenerative disorder, aneurological disorder, a mood disorder, a cardiovascular disease, ametabolic disorder, a respiratory disease, a drug sensitivity condition,an eye disease, an immunological disorder, or a hematological disease.3. The method of claim 1, wherein the differentiated cells are neuralstem cells, neurons, cardiomyocytes, hepatic stem cells, or hepatocytes.4. The method of claim 1, wherein the phenotype is apoptosis,intracellular calcium level, calcium flux, protein kinase activity,mitochondrial oxidative stress, enzyme activity, cell morphology,receptor activation, protein trafficking, intracellular proteinaggregation, organellar composition, motility, intercellularcommunication, protein expression, or gene expression.
 5. The method ofclaim 1, further comprising comparing a plurality of polymorphic allelespresent in the genome of the human induced pluripotent stem cell line toa plurality of polymorphic alleles present in the genome of a humanother than the human from which the induced pluripotent stem cell linewas generated.
 6. The method of claim 1, further comprising genotypingthe human induced pluripotent stem cell line for a plurality ofpolymorphisms.
 7. The method of claim 1, further comprising comparingthe genome sequence of the human induced pluripotent stem cell line, ora portion thereof, to the genome sequence, or a portion thereof, of ahuman other than the human from which the induced pluripotent stem cellline was generated.
 8. The method of claim 1, further comprisingsequencing the genome of the human induced pluripotent stem cell line.9. A method for assessing the risk of drug toxicity in a human subject,comprising: (i) generating iPS cells from one or more isolated somaticcells obtained from the human subject; (ii) differentiating the iPScells obtained in step (i) to obtain one or more isolated differentiatedcells; (iii) contacting one or more cells obtained in step (ii) with adose of a pharmacological agent; (iv) assaying the contacted one or moredifferentiated cells for toxicity; and (v) determining that there is alow risk for toxicity if the assay is negative for toxicity in thecontacted cells; or (vi) determining that there is a high risk oftoxicity if the assay is positive for toxicity in the contacted cells;wherein the differentiated cells comprise an exogenous Oct3/4 gene, anexogenous Sox2 gene and an exogenous Klf4 gene.
 10. A method forassessing the efficacy of a dose of a pharmacological agent in a humansubject, comprising the steps of: (i) generating iPS cells from one ormore isolated somatic cells obtained from the human subject; (ii)differentiating the iPS cells obtained in step (i) to obtain one or moreisolated differentiated cells; (iii) contacting one or more cellsobtained in step (ii) with the dose of the pharmacological agent; (iv)assaying the contacted one or more differentiated cells for a marker ofefficacy of the pharmacological agent; and (v) determining that the doseof the pharmacological agent is effective if the assay is positive forefficacy in the contacted cells; or (vi) determining that the dose ofthe pharmacological agent is not effective if the assay is negative forefficacy in the contacted cells; wherein the differentiated cellscomprise an exogenous Oct3/4 gene, an exogenous Sox2 gene and anexogenous Klf4 gene.
 11. A method for identifying an agent that isuseful as a drug, comprising the steps of: (i) contacting one or moreisolated cells differentiated from an induced pluripotent stem cell linewith a dose of a candidate agent; (ii) assaying the contacted one ormore differentiated cells for desired efficacy; and (iii) identifyingthe candidate agent as useful if the assay is positive for efficacy inthe contacted cells; or (iv) identifying the candidate agent as notuseful if the assay is negative for efficacy in the contacted cells;wherein the one or more cells differentiated from the inducedpluripotent stem cell line comprise an exogenous Oct3/4 gene, anexogenous Sox2 gene, and an exogenous Klf4 gene.